Cytidine-5-carboxamide modified nucleotide compositions and methods related thereto

ABSTRACT

Described herein are 5-position modified cytosine nucleotides and nucleosides as well as phosphoramidites and triphosphates derivatives thereof. Further provided are methods of making and using the same, and compositions and uses of the modified nucleosides as part of a nucleic acid molecule (e.g., aptamer).

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/917,056, filed Mar. 7, 2016. U.S. application Ser. No. 14/917,056 isa 35 U.S.C. § 371 national phase application of InternationalApplication Serial No. PCT/US2014/066328 (WO 2015/077292), filed Nov.19, 2014. International Application Serial No. PCT/US2014/066328 claimspriority under 35 U.S.C. § 119(e) to U.S. Provisional Application Ser.No. 61/907,274, filed on Nov. 21, 2013. The content of each of theseapplications is incorporated herein by reference in its entirety.

FIELD

The present disclosure relates generally to the field of nucleic acidchemistry, specifically to 5-position modified cytosine as well asphosphoramidites and triphosphates derivatives thereof. The presentdisclosure also relates to methods of making and using the same. Thedisclosure includes the use of the modified nucleosides as part of anoligonucleotide or an aptamer.

Incorporated by reference herein in its entirety is the Sequence Listingentitled “Sequences-0057-65PCT_ST25”, created Oct. 2, 2014, size of 2kilobytes.

BACKGROUND

Modified nucleosides have been used as therapeutic agents, diagnosticagents, and for incorporation into oligonucleotides to improve theirproperties (e.g., stability).

SELEX (Systematic Evolution of Ligands for EXponential Enrichment) is amethod for identifying oligonucleotides (referred to as “aptamers”) thatselectively bind target molecules. The SELEX process is described inU.S. Pat. No. 5,270,163, the contents of which are herein incorporatedby reference in their entirety. The SELEX method involves the selectionand identification of oligonucleotides from a random mixture ofoligonucleotides to achieve virtually any desired criterion of bindingaffinity and selectivity. By introducing specific types of modifiednucleosides to the oligonucleotides identified in the course of theSELEX process, the nuclease stability, net charge, hydrophilicity orlipophilicity may be altered to provide differences in the threedimensional structure and target binding capabilities of theoligonucleotides. Thus, different modified nucleosides provide theability to “tune” the desired properties of an oligonucleotide selectedin the course of SELEX.

Modified deoxyuridine nucleotides, bearing an N-substituted-carboxamidegroup at the 5-position, have proven to be valuable tools for improvingin vitro selection of protein-binding aptamers (SELEX process) (see,e.g., Gold et al., 2010; Hollenstein, 2012; and Imaizumi et al., 2013)and for post-SELEX optimization of binding and pharmacokineticproperties of the selected aptamers (see, e.g., Davies, et al., 2012;Lee et al., 2010; Kerr et al., 2000; and Gaballah et al., 2002). Thegeneral synthesis of uridine-5-carboxamides relied on a common activatedester intermediate, 5-(2,2,2-trifluoroethoxycarbonyl)-2′-deoxyuridine(1), which was originally reported by Matsuda and coworkers (see, e.g.Nomura et al., 1997). Treatment of this activated ester with variousprimary amines (1.2 eq., 60° C., 4 h) affords the corresponding5-(N-substituted-carboxamides). Matsuda also disclosed the analogousactivated ester in the cytidine series,N-acetyl-5-(2,2,2-trifluoroethoxycarbonyl)-2′-deoxycytidine (see, e.g.Nomura et al., 1996). However, this intermediate was less practicallyuseful for synthesis of cytidine-5-carboxamides due to the lability ofthe N-acetyl protecting group and the instability of theN-acetyl-5-iodo-cytidine synthetic precursors.

There continues to be a need for alternative composition for improvingoligonucleotide target binding agents, and further methods forsynthesizing such compositions. The present disclosure meets such needsby providing novel cytidine-5-carboxamide modified compositions.

SUMMARY

The present disclosure describes 5-position modified cytosine as well asphosphoramidites and triphosphates derivatives thereof, and to methodsof making and using the same.

In one aspect, the disclosure provides for a compound comprising thestructure shown in Formula I:

wherein

R is independently a —(CH₂)_(n)—, wherein n is an integer selected from0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

R^(X1) is independently selected from the group consisting of:

wherein * denotes the point of attachment of the R^(X1) group to the—(CH₂)_(n)— group; and wherein

R^(X4) is independently selected from the group consisting of asubstituted or unsubstituted branched or linear lower alkyl (C1-C20); ahydroxyl group; a halogen (F, Cl, Br, I); nitrile (CN); boronic acid(BO₂H₂); carboxylic acid (COOH); carboxylic acid ester (COOR^(X2));primary amide (CONH₂); secondary amide (CONHR^(X2)); tertiary amide(CONR^(X2)R^(X3)); sulfonamide (SO₂NH₂); N-alkylsulfonamide(SONHR^(X2));

R^(X2) and R^(X3) are independently, for each occurrence, selected fromthe group consisting of a substituted or unsubstituted branched orlinear lower alkyl (C1-C20); phenyl (C₆H₅); an R^(X4) substituted phenylring (R^(X4)C₆H₄), wherein R^(X4) is defined above; a carboxylic acid(COOH); a carboxylic acid ester (COOR^(X5)), wherein R^(X5) is abranched or linear lower alkyl (C1-C20); and cycloalkyl, wherein R^(X2)and R^(X3) together form a substituted or unsubstituted 5 or 6 memberedring;

X is independently selected from the group consisting of —H, —OH, —OMe,—O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃, —NH₂ and -azido;

R′ is independently selected from the group consisting of a —H, —OAc;—OBz; —P(NiPr₂)(OCH₂CH₂CN); and —OSiMe₂tBu;

R″ is independently selected from the group consisting of a hydrogen,4,4′-dimethoxytrityl (DMT) and triphosphate(—P(O)(OH)—O—P(O)(OH)—O—P(O)(OH)₂) or a salt thereof;

Z is independently selected from the group consisting of a —H, asubstituted or unsubstituted branched or linear lower alkyl (C1-C4);

and salts thereof;

with the following exceptions:

when n=4, then R^(X1) cannot be H;

when n=3, then R^(X1) cannot be CH₃;

when n=0, then R^(X1) cannot be —CH(CH₃)₂; and

when n=2, and R^(X1) is

and R^(X4) is hydroxyl then R^(X1) cannot be

In related aspect n is an integer selected from 1, 2 or 3.

In related aspect, R^(X1) is selected from the group consisting of:

wherein

* denotes the point of attachment of the R^(X1) group to the —(CH₂)_(n)—group; and

Z is independently selected from the group consisting of a —H, asubstituted or unsubstituted branched or linear lower alkyl (C1-C4).

In related aspect, R^(X4) is independently selected from the groupconsisting of a branched or linear lower alkyl (C1-C6); —OH; —F andcarboxylic acid (COOH).

In related aspect, X is independently selected from the group consistingof —H, —OH, —OMe and —F.

In related aspect, R′ is selected from the group consisting of a —H,—OAc and —P(NiPr₂)(OCH₂CH₂CN).

In related aspect, R″ is a triphosphate(—P(O)(OH)—O—P(O)(OH)—O—P(O)(OH)₂).

In another aspect, the disclosure provides for a compound comprising thestructure selected from the group consisting of Formulas II (BndC), III(PEdC), IV (PPdC), V (NapdC), VI (2NapdC), VII (NEdC) and VIII (2NEdC):

wherein

X is independently selected from the group consisting of —H, —OH, —OMe,—O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃, —NH₂ and -azido.

In another aspect, the disclosure provides for a nucleic acid moleculecomprising any one of the compounds described above.

In a related aspect, the nucleic acid molecule comprises RNA, DNA or acombination thereof.

In a related aspect, the nucleic acid molecule is from 15 to 100nucleotides in length.

In a related aspect, the nucleic acid molecule is an aptamer.

In a related aspect, at least one additional nucleotide of the nucleicacid molecule comprises a chemical modification selected from the groupconsisting of a 2′-position sugar modification including but not limitedto, a 2′-amino (2′-NH₂), 2′-fluoro (2′-F), 2′-O-methyl (2′-OMe),2′-O-ethyl (2′-OEt), 2′-O-propyl (2′-OPr), 2′-O—CH₂CH₂OCH₃ and azido.

In another aspect, the disclosure provides for a nucleic acid moleculecomprising a compound comprising the structure shown in Formula IA:

wherein

R is independently a —(CH₂)_(n)—, wherein n is an integer selected from0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

R^(X1) is independently selected from the group consisting of:

wherein, * denotes the point of attachment of the R^(X1) group to the—(CH₂)_(n)— group; and wherein,

R^(X4) is independently selected from the group consisting of a branchedor linear lower alkyl (C1-C20); a hydroxyl group; halogen (F, Cl, Br,I); nitrile (CN); boronic acid (BO₂H₂); carboxylic acid (COOH);carboxylic acid ester (COOR^(X2)); primary amide (CONH₂); secondaryamide (CONHR^(X2)); tertiary amide (CONR^(X2)R^(X3)); sulfonamide(SO₂NH₂); N-alkylsulfonamide (SONHR^(X2));

R^(X2) and R^(X3) are independently, for each occurrence, selected fromthe group consisting of a branched or linear lower alkyl (C1-C20);phenyl (C₆H₅); an R^(X4) substituted phenyl ring (R^(X4)C₆H₄), whereinR^(X4) is defined above; a carboxylic acid (COOH); a carboxylic acidester (COOR^(X5)), wherein R^(X5) is a branched or linear lower alkyl(C1-C20); and cycloalkyl, wherein R^(X2) and R^(X3) together form asubstituted or unsubstituted 5 or 6 membered ring;

X is independently selected from the group consisting of —H, —OH, —OMe,—O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃, NH₂ and -azido;

Z is independently selected from the group consisting of a —H, asubstituted or unsubstituted C(1-4)alkyl;

and salts thereof;

with the following exceptions:

when n=4, then R^(X1) cannot be H;

when n=3, then R^(X1) cannot be CH₃;

when n=0, then R^(X1) cannot be —CH(CH₃)₂; and

when n=2, and R^(X1) is

and R^(X4) is hydroxyl then R^(X1) cannot be

In a related aspect, n is 1, 2 or 3.

In a related aspect, R^(X1) is selected from the group consisting of:

wherein,

* denotes the point of attachment of the R^(X1) group to the —(CH₂)_(n)—group; and

Z is independently selected from the group consisting of a —H, asubstituted or unsubstituted C(1-4)alkyl.

In a related aspect, R^(X4) is independently selected from the groupconsisting of a branched or linear lower alkyl (C1-C6); a —OH; a —F andcarboxylic acid (COOH).

In a related aspect, X is independently selected from the groupconsisting of —H, —OH, —OMe and —F.

In a related aspect, R′ is selected from the group consisting of a —H,—OAc and —P(NiPr₂)(OCH₂CH₂CN).

In a related aspect, R″ is a triphosphate(—P(O)(OH)—O—P(O)(OH)—O—P(O)(OH)₂).

In a related aspect, the nucleic acid molecule comprises DNA, RNA or acombination thereof.

In a related aspect, the nucleic acid molecule is from 15 to 100nucleotides in length.

In a related aspect, the nucleic acid molecule is an aptamer.

In a related aspect, at least one additional nucleotide of the nucleicacid molecule comprises a chemical modification selected from the groupconsisting of a 2′-position sugar modification independently selectedfrom the group consisting of 2′-amino (2′-NH₂), 2′-fluoro (2′-F),2′-O-methyl (2′-OMe), 2′-O-ethyl (2′-OEt), 2′-O-propyl (2′-OPr),2′-O—CH₂CH₂OCH₃ and azido.

In a related aspect, the nucleic acid molecule further comprises amodification selected from the group consisting of a backbonemodification, a 3′ cap, a 5′ cap and a combination thereof.

In a related aspect, the compound comprises the structure selected fromthe group consisting of Formulas IIA, IIIA, IVA, VA, VIA, VIIA andVIIIA:

wherein

X is independently selected from the group consisting of —H, —OH, —OMe,—O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃, NH₂ and -azido.

In another aspect, the disclosure provides for a method for making acompound having Formula I:

wherein

R is independently a —(CH₂)_(n)—, wherein n is an integer selected from0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

R^(X1) is independently selected from the group consisting of:

wherein, * denotes the point of attachment of the R^(X1) group to the—(CH₂)_(n)— group; and wherein,

R^(X4) is independently selected from the group consisting of a branchedor linear lower alkyl (C1-C20); a hydroxyl group; halogen (F, Cl, Br,I); nitrile (CN); boronic acid (BO₂H₂); carboxylic acid (COOH);carboxylic acid ester (COOR^(X2)); primary amide (CONH₂); secondaryamide (CONHR^(X2)); tertiary amide (CONR^(X2)R^(X3)); sulfonamide(SO₂NH₂); N-alkylsulfonamide (SONHR^(X2));

R^(X2) and R^(X3) are independently, for each occurrence, selected fromthe group consisting of a branched or linear lower alkyl (C1-C20);phenyl (C₆H₅); an R^(X4) substituted phenyl ring (R^(X4)C₆H₄), whereinR^(X4) is defined above; a carboxylic acid (COOH); a carboxylic acidester (COOR^(X5)), wherein R^(X5) is a branched or linear lower alkyl(C1-C20); and cycloalkyl, wherein R^(X2) and R^(X3) together form asubstituted or unsubstituted 5 or 6 membered ring;

X is independently selected from the group consisting of —H, —OH, —OMe,—O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃, NH₂ and -azido;

Z is independently selected from the group consisting of a —H, asubstituted or unsubstituted C(1-4)alkyl;

the method comprising providing a compound having Formula IX

wherein,

R^(X6) is an iodine or bromine group;

R^(X7) and R^(X8) are independently, for each occurrence, a hydrogen ora protecting group;

X is independently selected from the group consisting of —H, —OH, —OMe,—O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃, NH₂ and -azido; and

transforming the compound having Formula IX by a palladium(0) catalyzedreaction in the presence of R^(X1)—R—NH₂, carbon monoxide and a solvent;and

isolating the compound having Formula I.

In a related aspect, R^(X6) is an iodine group.

In a related aspect, R^(X7) and R^(X8) are a hydrogen.

In a related aspect, X is selected from the group consisting of a —H,—OMe and —F.

In a related aspect, n is 1, 2 or 3.

In a related aspect, R^(X1) is selected from the group consisting of:

wherein,

* denotes the point of attachment of the R^(X1) group to the —(CH₂)_(n)—group; and

Z is independently selected from the group consisting of a —H, asubstituted or unsubstituted C(1-4)alkyl.

In a related aspect, R^(X4) is independently selected from the groupconsisting of a branched or linear lower alkyl (C1-C6); a —OH; a —F andcarboxylic acid (COOH).

In a related aspect, R′ is selected from the group consisting of a —H,—OAc and —P(NiPr₂)(OCH₂CH₂CN).

In a related aspect, R″ is a hydrogen or triphosphate(—P(O)(OH)—O—P(O)(OH)—O—P(O)(OH)₂).

In a related aspect, the protecting group is selected from the groupconsisting of triphenylmethyl, p-anisyldiphenylmethyl,di-p-anisyldiphenylmethyl, p-dimethoxy trityltrityl, formyl,t-butyloxycarbonyl, benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl,4-chlorobenzoyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl,furfurylcarbonyl, t-amyloxycarbonyl, adamantyloxycarbonyl,2-phenylpropyl-(2)-oxycarbonyl, 2-(4-biphenyl)propyl-(2)-oxycarbonyl,2-nitrophenylsulfenyl and diphenylphosphinyl.

In a related aspect, the solvent is selected from the group consistingof dimethylformamide (DMF), dichloromethane (DCM), tetrahydrofuran(THF), ethyl acetate, acetone, acetronitrile (MeCN), dimethyl sulfoxide(DMSO) and propylene carbonate.

In another aspect, the disclosure provides for a method for making acompound having a formula selected from the group consisting of FormulasII, III, IV, V, VI, VII and VIII

wherein

X is independently selected from the group consisting of —H, —OH, —OMe,—O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃, NH₂ and -azido;

the method comprising providing a compound having Formula IX

wherein,

R^(X6) is an iodine or bromine group;

R^(X7) and R^(X8) are independently, for each occurrence, a hydrogen orprotecting group;

X is independently selected from the group consisting of —H, —OH, —OMe,—O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃ and -azido; and

transforming the compound having Formula IX by a palladium(0) catalyzedreaction in the presence of R^(X1)—R—NH₂, carbon monoxide and a solvent;and

isolating the compound having the formula selected from the groupconsisting of Formulas II, III and IV.

In a related aspect, R^(X6) is an iodine group.

In a related aspect, R^(X7) and R^(X8) are hydrogen.

In a related aspect, X is selected from the group consisting of a —H,—OMe and —F.

In a related aspect, the protecting group is selected from the groupconsisting of triphenylmethyl, p-anisyldiphenylmethyl,di-p-anisyldiphenylmethyl, p-dimethoxy trityltrityl, formyl,t-butyloxycarbonyl, benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl,4-chlorobenzoyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl,furfurylcarbonyl, t-amyloxycarbonyl, adamantyloxycarbonyl,2-phenylpropyl-(2)-oxycarbonyl, 2-(4-biphenyl)propyl-(2)-oxycarbonyl,2-nitrophenylsulfenyl and diphenylphosphinyl.

In a related aspect, the solvent is selected from the group consistingof dimethylformamide (DMF), dichloromethane (DCM), tetrahydrofuran(THF), ethyl acetate, acetone, acetronitrile (MeCN), dimethyl sulfoxide(DMSO) and propylene carbonate.

The present disclosure further provide for a method for selecting anucleic acid aptamer having binding affinity for a target moleculecomprising: (a) contacting a candidate mixture with the target, whereinthe candidate mixture comprises modified nucleic aptamers in which one,several or all pyrimidines in at least one, or each, nucleic acidaptamer of the candidate mixture comprises a compound described herein(5-position modified cytosine), and wherein nucleic acid aptamers havingbinding affinity for the target molecule form nucleic acidaptamer-target molecule complexes; (b) partitioning the nucleic acidaptamer-target molecule complexes from the candidate mixture; (c)dissociating the nucleic acid aptamer-target molecule complexes togenerate free nucleic acid aptamers; (d) amplifying the free nucleicacid aptamers to yield nucleic acid aptamers having an increaseddissociation half-life from the target molecule relative to othernucleic acids in the candidate mixture; (e) identifying at least onenucleic acid aptamer, wherein the nucleic acid aptamer has bindingaffinity for the target molecule.

In another aspect, steps a) through d) are repeated with the mixture ofnucleic acid aptamers enriched in nucleic acid sequences capable ofbinding to the target molecule and have a slow off-rate when bound tothe target molecule to further enrich for nucleic acid sequences thatare capable of binding to the target molecule and have a slow off-ratewhen bound to the target molecule.

In another aspect, the rate of dissociation of the slow off-rate nucleicacid aptamer is from about 2 minutes to about 360 minutes (or from about2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 45, 60, 75, 90, 105, 120,150, 180, 210, 240, 270, 300, 330 or 360 minutes).

In another aspect the rate of dissociation of the slow off-rate nucleicacid aptamer is greater than or equal to about 2 minutes (or greaterthan about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 45, 60, 75, 90,105, 120, 150, 180, 210, 240, 270, 300, 330 or 360 minutes).

In another aspect the target molecule is a protein or a peptide.

In another aspect the target molecule is selected from the groupconsisting of a PSCK9 protein, a PSMA protein, ERBB2 protein and a ERBB3protein.

In another aspect the at least one nucleic acid aptamer is capable ofbinding the target molecule with an equilibrium binding constant (K_(d))of at less than 100 nM, or from about 0.1 nM to about 100 nM (or from0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or100 nM).

In another aspect, the methods described herein further compriseexposing the candidate mixture to a slow off-rate enrichment process.

In another aspect, the slow off-rate enrichment process is performedprior to step (b). In a related aspect, the slow off-rate enrichmentprocess is selected from the group consisting of adding a competitormolecule, a dilution step, a combination of adding a competitor moleculefollowed by a dilution step, a combination of a dilution step followedby a adding a competitor molecule, and a combination of simultaneouslyadding a competitor molecule and a dilution step.

In yet another related aspect, the competitor molecule is a polyanion.In another aspect, the competitor molecule is selected from the groupconsisting of an oligonucleotide, dNTPs, heparin and dextran sulfate.

The foregoing and other objects, features, and advantages of theinvention will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying FIGURES.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a polyacrylamide gel image of a primer extension assay withDNTP's as described in the Materials and Methods section of theExamples. Lane 1: dAdGdT (5% full length); Lane 2: dAdGdTdC (100% fulllength); Lane 3: dAdGdT+9a (119% full length); Lane 4: dAdGdT+9b (113%full length); Lane 5: dAdGdT+9c (120% full length); Lane 6: 20/200 DNALadder. With reference to this FIGURE it can be seen that all threemodified cytidine triphosphates were incorporated at least asefficiently as natural, unmodified 2′-deoxycytidine in this assay.

DETAILED DESCRIPTION

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular termswherein, * denotes the point of attachment of the R^(X1) group to the—(CH₂)_(n)— group; and wherein, * denotes the point of attachment of theR^(X1) group to the —(CH₂)_(n)— group; and “a,” “an,” and “the” includeplural referents unless context clearly indicates otherwise. “ComprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description.

Further, ranges provided herein are understood to be shorthand for allof the values within the range. For example, a range of 1 to 50 isunderstood to include any number, combination of numbers, or sub-rangefrom the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or50 (as well as fractions thereof unless the context clearly dictatesotherwise). Any concentration range, percentage range, ratio range, orinteger range is to be understood to include the value of any integerwithin the recited range and, when appropriate, fractions thereof (suchas one tenth and one hundredth of an integer), unless otherwiseindicated. Also, any number range recited herein relating to anyphysical feature, such as polymer subunits, size or thickness, are to beunderstood to include any integer within the recited range, unlessotherwise indicated. As used herein, “about” or “consisting essentiallyof” mean±20% of the indicated range, value, or structure, unlessotherwise indicated. As used herein, the terms “include” and “comprise”are open ended and are used synonymously. It should be understood thatthe terms “a” and “an” as used herein refer to “one or more” of theenumerated components. The use of the alternative (e.g., “or”) should beunderstood to mean either one, both, or any combination thereof of thealternatives

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of the present disclosure,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

As used herein, the term “nucleotide” refers to a ribonucleotide or adeoxyribonucleotide, or a modified form thereof, as well as an analogthereof. Nucleotides include species that include purines (e.g.,adenine, hypoxanthine, guanine, and their derivatives and analogs) aswell as pyrimidines (e.g., cytosine, uracil, thymine, and theirderivatives and analogs).

As used herein, the term “C-5 modified carboxamidecytidine” or“cytidine-5-carboxamide” refers to a cytidine with a carboxyamide(—C(O)NH—) modification at the C-5 position of the cytidine including,but not limited to, those moieties (R^(X1)) illustrated herein. ExampleC-5 modified carboxamidecytidine include, but are not limited to,5-(N-benzylcarboxamide)-2′-deoxycytidine (referred to as “BndC” andshown below as Formula (II);5-(N-2-phenylethylcarboxamide)-2′-deoxycytidine (referred to as “PEdC”and shown below as Formula (III);5-(N-3-phenylpropylcarboxamide)-2′-deoxycytidine (referred to as “PPdC”and shown below as Formula (IV);5-(N-1-naphthylmethylcarboxamide)-2′-deoxycytidine (referred to as“NapdC” and shown below as Formula (V);5-(N-2-naphthylmethylcarboxamide)-2′-deoxycytidine (referred to as“2NapdC” and shown below as Formula (VI);5-(N-1-naphthyl-2-ethylcarboxamide)-2′-deoxycytidine (referred to as“NEdC” and shown below as Formula (VII); and5-(N-2-naphthyl-2-ethylcarboxamide)-2′-deoxycytidine (referred to as“2NEdC” and shown below as Formula (VIII):

Chemical modifications of the C-5 modified cytidines described hereincan also be combined with, singly or in any combination, 2′-positionsugar modifications, modifications at exocyclic amines, and substitutionof 4-thiocytidine and the like.

Salts

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge et al. (1977) “PharmaceuticallyAcceptable Salts” J. Pharm. Sci. 66:1-19.

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g., —COOH may be —COO⁻), then a salt may be formedwith a suitable cation. Examples of suitable inorganic cations include,but are not limited to, alkali metal ions such as Na⁺ and K⁺, alkalineearth cations such as Ca²⁺ and Mg²⁺, and other cations such as Al⁺³.Examples of suitable organic cations include, but are not limited to,ammonium ion (i.e., NH₄ ⁺) and substituted ammonium ions (e.g.,NH₃R^(X+), NH₂R^(X) ₂ ⁺, NHR^(X) ₃ ⁺, NR^(X) ₄ ⁺). Examples of somesuitable substituted ammonium ions are those derived from: ethylamine,diethylamine, dicyclohexylamine, triethylamine, butylamine,ethylenediamine, ethanolamine, diethanolamine, piperizine, benzylamine,phenylbenzylamine, choline, meglumine, and tromethamine, as well asamino acids, such as lysine and arginine. An example of a commonquaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g., —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examplesof suitable polymeric organic anions include, but are not limited to,those derived from the following polymeric acids: tannic acid,carboxymethyl cellulose.

Unless otherwise specified, a reference to a particular compound alsoincludes salt forms thereof.

Preparation of Oligonucleotides

In one aspect, the instant disclosure provides methods for using themodified nucleosides described herein, either alone or in combinationwith other modified nucleosides and/or naturally occurring nucleosides,to prepare modified oligonucleotides. The automated synthesis ofoligodeoxynucleosides is routine practice in many laboratories (seee.g., Matteucci, M. D. and Caruthers, M. H., (1990) J. Am. Chem. Soc.,103:3185-3191, the contents of which are hereby incorporated byreference in their entirety). Synthesis of oligoribonucleosides is alsowell known (see e.g. Scaringe, S. A., et al., (1990) Nucleic Acids Res.18:5433-5441, the contents of which are hereby incorporated by referencein their entirety). As noted herein, the phosphoramidites are useful forincorporation of the modified nucleoside into an oligonucleotide bychemical synthesis, and the triphosphates are useful for incorporationof the modified nucleoside into an oligonucleotide by enzymaticsynthesis. (See e.g., Vaught, J. D. et al. (2004) J. Am. Chem. Soc.,126:11231-11237; Vaught, J. V., et al. (2010) J. Am. Chem. Soc. 132,4141-4151; Gait, M. J. “Oligonucleotide Synthesis a practical approach”(1984) IRL Press (Oxford, UK); Herdewijn, P. “Oligonucleotide Synthesis”(2005) (Humana Press, Totowa, N.J. (each of which is incorporated hereinby reference in its entirety).

As used herein, the terms “modify,” “modified,” “modification,” and anyvariations thereof, when used in reference to an oligonucleotide, meansthat at least one of the four constituent nucleotide bases (i.e., A, G,T/U, and C) of the oligonucleotide is an analog or ester of a naturallyoccurring nucleotide. In some embodiments, the modified nucleotideconfers nuclease resistance to the oligonucleotide. Additionalmodifications can include backbone modifications, methylations, unusualbase-pairing combinations such as the isobases isocytidine andisoguanidine, and the like. Modifications can also include 3′ and 5′modifications, such as capping. Other modifications can includesubstitution of one or more of the naturally occurring nucleotides withan analog, internucleotide modifications such as, for example, thosewith uncharged linkages (e.g., methyl phosphonates, phosphotriesters,phosphoamidates, carbamates, etc.) and those with charged linkages(e.g., phosphorothioates, phosphorodithioates, etc.), those withintercalators (e.g., acridine, psoralen, etc.), those containingchelators (e.g., metals, radioactive metals, boron, oxidative metals,etc.), those containing alkylators, and those with modified linkages(e.g., alpha anomeric nucleic acids, etc.). Further, any of the hydroxylgroups ordinarily present on the sugar of a nucleotide may be replacedby a phosphonate group or a phosphate group; protected by standardprotecting groups; or activated to prepare additional linkages toadditional nucleotides or to a solid support. The 5′ and 3′ terminal OHgroups can be phosphorylated or substituted with amines, organic cappinggroup moieties of from about 1 to about 20 carbon atoms, polyethyleneglycol (PEG) polymers in one embodiment ranging from about 10 to about80 kDa, PEG polymers in another embodiment ranging from about 20 toabout 60 kDa, or other hydrophilic or hydrophobic biological orsynthetic polymers.

Polynucleotides can also contain analogous forms of ribose ordeoxyribose sugars that are generally known in the art, including2′-O-methyl, 2′-O-allyl, 2′-O-ethyl, 2′-O-propyl, 2′-O—CH₂CH₂OCH₃,2′-fluoro, 2′-NH₂ or 2′-azido, carbocyclic sugar analogs, α-anomericsugars, epimeric sugars such as arabinose, xyloses or lyxoses, pyranosesugars, furanose sugars, sedoheptuloses, acyclic analogs and abasicnucleoside analogs such as methyl riboside. As noted herein, one or morephosphodiester linkages may be replaced by alternative linking groups.These alternative linking groups include embodiments wherein phosphateis replaced by P(O)S (“thioate”), P(S)S (“dithioate”), (O)NR^(X) ₂(“amidate”), P(O) R^(X), P(O)OR^(X)′, CO or CH₂ (“formacetal”), in whicheach R^(X) or R^(X)′ are independently H or substituted or unsubstitutedalkyl (C1-C20) optionally containing an ether (—O—) linkage, aryl,alkenyl, cycloalky, cycloalkenyl or araldyl. Not all linkages in apolynucleotide need be identical. Substitution of analogous forms ofsugars, purines, and pyrimidines can be advantageous in designing afinal product, as can alternative backbone structures like a polyamidebackbone, for example.

Polynucleotides can also contain analogous forms of carbocyclic sugaranalogs, α-anomeric sugars, epimeric sugars such as arabinose, xylosesor lyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and abasic nucleoside analogs such as methyl riboside.

If present, a modification to the nucleotide structure can be impartedbefore or after assembly of a polymer. A sequence of nucleotides can beinterrupted by non-nucleotide components. A polynucleotide can befurther modified after polymerization, such as by conjugation with alabeling component.

As used herein, “nucleic acid,” “oligonucleotide,” and “polynucleotide”are used interchangeably to refer to a polymer of nucleotides andinclude DNA, RNA, DNA/RNA hybrids and modifications of these kinds ofnucleic acids, oligonucleotides and polynucleotides, wherein theattachment of various entities or moieties to the nucleotide units atany position are included. The terms “polynucleotide,”“oligonucleotide,” and “nucleic acid” include double- or single-strandedmolecules as well as triple-helical molecules. Nucleic acid,oligonucleotide, and polynucleotide are broader terms than the termaptamer and, thus, the terms nucleic acid, oligonucleotide, andpolynucleotide include polymers of nucleotides that are aptamers but theterms nucleic acid, oligonucleotide, and polynucleotide are not limitedto aptamers.

In certain embodiments, the disclosure provides for a method for makinga nucleic acid molecule comprising a compound having Formula I:

wherein

R is independently a —(CH₂)_(n)—, wherein n is an integer selected from0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

R^(X1) is independently selected from the group consisting of

wherein, * denotes the point of attachment of the R^(X1) group to the—(CH₂)_(n)— group; and wherein,

R^(X4) is independently selected from the group consisting of a branchedor linear lower alkyl (C1-C20); a hydroxyl group; a halogen (F, Cl, Br,I); nitrile (CN); boronic acid (BO₂H₂); carboxylic acid (COOH);carboxylic acid ester (COOR^(X2)); primary amide (CONH₂); secondaryamide (CONHR^(X2)); tertiary amide (CONR^(X2)R^(X3)); sulfonamide(SO₂NH₂); N-alkylsulfonamide (SONHR^(X2));

R^(X2) and R^(X3) are independently, for each occurrence, selected fromthe group consisting of a branched or linear lower alkyl (C1-C20);phenyl (C₆H₅); an R^(X4) substituted phenyl ring (R^(X4)C₆H₄), whereinR^(X4) is defined above; a carboxylic acid (COOH); a carboxylic acidester (COOR^(X5)), wherein R^(X5) is a branched or linear lower alkyl(C1-C20); and cycloalkyl, wherein R^(X2) and R^(X3) together form asubstituted or unsubstituted 5 or 6 membered ring;

X is independently selected from the group consisting of —H, —OH, —OMe,—O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃, NH₂ and -azido;

R′ is independently selected from the group consisting of a —H, —OAc;—OBz; —P(NiPr₂)(OCH₂CH₂CN); and —OSiMe₂tBu;

R″ is independently selected from the group consisting of a hydrogen,4,4′-dimethoxytrityl (DMT) and triphosphate(—P(O)(OH)—O—P(O)(OH)—O—P(O)(OH)₂) or a salt thereof;

Z is independently selected from the group consisting of a —H, asubstituted or unsubstituted C(1-4)alkyl;

and salts thereof, the method comprising synthesizing a nucleic acidmolecule having a plurality of nucleotides and at least one compoundhaving Formula I.

In certain embodiments, the disclosure provides for method for making anucleic acid molecule comprising a compound having a formula selectedfrom the group consisting of Formulas II, III, IV, V, VI, VII and VIII:

wherein

X is independently selected from the group consisting of —H, —OH, —OMe,—O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃, NH₂ and -azido;

the method comprising synthesizing a nucleic acid molecule having aplurality of nucleotides and at least one compound having the formulaselected from the group consisting of Formulas II, III and IV.

As used herein, the term “at least one nucleotide” when referring tomodifications of a nucleic acid, refers to one, several, or allnucleotides in the nucleic acid, indicating that any or all occurrencesof any or all of A, C, T, G or U in a nucleic acid may be modified ornot.

In other aspects, the instant disclosure methods for using the modifiednucleosides described herein, either alone or in combination with othermodified nucleosides and/or naturally occurring nucleosides, to prepareaptamers and SOMAmers (described herein). In specific embodiments, theaptamers and SOMAmers are prepared using the general SELEX or improvedSELEX process as described below.

As used herein, “nucleic acid ligand,” “aptamer,” “SOMAmer,” and “clone”are used interchangeably to refer to a non-naturally occurring nucleicacid that has a desirable action on a target molecule. A desirableaction includes, but is not limited to, binding of the target,catalytically changing the target, reacting with the target in a waythat modifies or alters the target or the functional activity of thetarget, covalently attaching to the target (as in a suicide inhibitor),and facilitating the reaction between the target and another molecule.In one embodiment, the action is specific binding affinity for a targetmolecule, such target molecule being a three dimensional chemicalstructure other than a polynucleotide that binds to the nucleic acidligand through a mechanism which is independent of Watson/Crick basepairing or triple helix formation, wherein the aptamer is not a nucleicacid having the known physiological function of being bound by thetarget molecule. Aptamers to a given target include nucleic acids thatare identified from a candidate mixture of nucleic acids, where theaptamer is a ligand of the target, by a method comprising: (a)contacting the candidate mixture with the target, wherein nucleic acidshaving an increased affinity to the target relative to other nucleicacids in the candidate mixture can be partitioned from the remainder ofthe candidate mixture; (b) partitioning the increased affinity nucleicacids from the remainder of the candidate mixture; and (c) amplifyingthe increased affinity nucleic acids to yield a ligand-enriched mixtureof nucleic acids, whereby aptamers of the target molecule areidentified. It is recognized that affinity interactions are a matter ofdegree; however, in this context, the “specific binding affinity” of anaptamer for its target means that the aptamer binds to its targetgenerally with a much higher degree of affinity than it binds to other,non-target, components in a mixture or sample. An “aptamer,” “SOMAmer,”or “nucleic acid ligand” is a set of copies of one type or species ofnucleic acid molecule that has a particular nucleotide sequence. Anaptamer can include any suitable number of nucleotides. “Aptamers” referto more than one such set of molecules. Different aptamers can haveeither the same or different numbers of nucleotides. Aptamers may be DNAor RNA and may be single stranded, double stranded, or contain doublestranded or triple stranded regions.

As used herein, a “SOMAmer” or Slow Off-Rate Modified Aptamer refers toan aptamer having improved off-rate characteristics. SOMAmers can begenerated using the improved SELEX methods described in U.S. Pat. No.7,947,447, entitled “Method for Generating Aptamers with ImprovedOff-Rates.”

As used herein, “protein” is used synonymously with “peptide,”“polypeptide,” or “peptide fragment.” A “purified” polypeptide, protein,peptide, or peptide fragment is substantially free of cellular materialor other contaminating proteins from the cell, tissue, or cell-freesource from which the amino acid sequence is obtained, or substantiallyfree from chemical precursors or other chemicals when chemicallysynthesized.

The SELEX Method

The terms “SELEX” and “SELEX process” are used interchangeably herein torefer generally to a combination of (1) the selection of nucleic acidsthat interact with a target molecule in a desirable manner, for examplebinding with high affinity to a protein, with (2) the amplification ofthose selected nucleic acids. The SELEX process can be used to identifyaptamers with high affinity to a specific target molecule or biomarker.

SELEX generally includes preparing a candidate mixture of nucleic acids,binding of the candidate mixture to the desired target molecule to forman affinity complex, separating the affinity complexes from the unboundcandidate nucleic acids, separating and isolating the nucleic acid fromthe affinity complex, purifying the nucleic acid, and identifying aspecific aptamer sequence. The process may include multiple rounds tofurther refine the affinity of the selected aptamer. The process caninclude amplification steps at one or more points in the process. See,e.g., U.S. Pat. No. 5,475,096, entitled “Nucleic Acid Ligands.” TheSELEX process can be used to generate an aptamer that covalently bindsits target as well as an aptamer that non-covalently binds its target.See, e.g., U.S. Pat. No. 5,705,337 entitled “Systematic Evolution ofNucleic Acid Ligands by Exponential Enrichment: Chemi-SELEX.”

The SELEX process can be used to identify high-affinity aptamerscontaining modified nucleotides that confer improved characteristics onthe aptamer, such as, for example, improved in vivo stability orimproved delivery characteristics. Examples of such modificationsinclude chemical substitutions at the ribose and/or phosphate and/orbase positions. SELEX process-identified aptamers containing modifiednucleotides are described in U.S. Pat. No. 5,660,985, entitled “HighAffinity Nucleic Acid Ligands Containing Modified Nucleotides,” whichdescribes oligonucleotides containing nucleotide derivatives chemicallymodified at the 5′- and 2′-positions of pyrimidines. U.S. Pat. No.5,580,737, see supra, describes highly specific aptamers containing oneor more nucleotides modified with 2′-amino (2′-NH₂), 2′-fluoro (2′-F),and/or 2′-O-methyl (2′-OMe). See also, U.S. Patent ApplicationPublication No. 20090098549, entitled “SELEX and PHOTOSELEX,” whichdescribes nucleic acid libraries having expanded physical and chemicalproperties and their use in SELEX and photoSELEX.

SELEX can also be used to identify aptamers that have desirable off-ratecharacteristics. See U.S. Pat. No. 7,947,447, entitled “Method forGenerating Aptamers with Improved Off-Rates,” which is incorporatedherein by reference in its entirety, describes improved SELEX methodsfor generating aptamers that can bind to target molecules. Methods forproducing aptamers and photoaptamers having slower rates of dissociationfrom their respective target molecules are described. The methodsinvolve contacting the candidate mixture with the target molecule,allowing the formation of nucleic acid-target complexes to occur, andperforming a slow off-rate enrichment process wherein nucleicacid-target complexes with fast dissociation rates dissociate and do notreform, while complexes with slow dissociation rates remain intact.Additionally, the methods include the use of modified nucleotides in theproduction of candidate nucleic acid mixtures to generate aptamers withimproved off-rate performance (see U.S. Pat. No. 8,409,795, entitled“SELEX and PhotoSELEX”). (See also U.S. Pat. No. 7,855,054 and U.S.Patent Publication No. 20070166740). Each of these applications isincorporated herein by reference in its entirety.

“Target” or “target molecule” or “target” refers herein to any compoundupon which a nucleic acid can act in a desirable manner. A targetmolecule can be a protein, peptide, nucleic acid, carbohydrate, lipid,polysaccharide, glycoprotein, hormone, receptor, antigen, antibody,virus, pathogen, toxic substance, substrate, metabolite, transitionstate analog, cofactor, inhibitor, drug, dye, nutrient, growth factor,cell, tissue, any portion or fragment of any of the foregoing, etc.,without limitation. Virtually any chemical or biological effector may bea suitable target. Molecules of any size can serve as targets. A targetcan also be modified in certain ways to enhance the likelihood orstrength of an interaction between the target and the nucleic acid. Atarget can also include any minor variation of a particular compound ormolecule, such as, in the case of a protein, for example, minorvariations in amino acid sequence, disulfide bond formation,glycosylation, lipidation, acetylation, phosphorylation, or any othermanipulation or modification, such as conjugation with a labelingcomponent, which does not substantially alter the identity of themolecule. A “target molecule” or “target” is a set of copies of one typeor species of molecule or multimolecular structure that is capable ofbinding to an aptamer. “Target molecules” or “targets” refer to morethan one such set of molecules. Embodiments of the SELEX process inwhich the target is a peptide are described in U.S. Pat. No. 6,376,190,entitled “Modified SELEX Processes Without Purified Protein.”

As used herein, “competitor molecule” and “competitor” are usedinterchangeably to refer to any molecule that can form a non-specificcomplex with a non-target molecule. In this context, non-targetmolecules include free aptamers, where, for example, a competitor can beused to inhibit the aptamer from binding (rebinding), non-specifically,to another non-target molecule. A “competitor molecule” or “competitor”is a set of copies of one type or species of molecule. “Competitormolecules” or “competitors” refer to more than one such set ofmolecules. Competitor molecules include, but are not limited tooligonucleotides, polyanions (e.g., heparin, herring sperm DNA, salmonsperm DNA, tRNA, dextran sulfate, polydextran, abasic phosphodiesterpolymers, dNTPs, and pyrophosphate). In various embodiments, acombination of one or more competitor can be used.

As used herein, “non-specific complex” refers to a non-covalentassociation between two or more molecules other than an aptamer and itstarget molecule. A non-specific complex represents an interactionbetween classes of molecules. Non-specific complexes include complexesformed between an aptamer and a non-target molecule, a competitor and anon-target molecule, a competitor and a target molecule, and a targetmolecule and a non-target molecule.

As used herein, the term “slow off-rate enrichment process” refers to aprocess of altering the relative concentrations of certain components ofa candidate mixture such that the relative concentration of aptameraffinity complexes having slow dissociation rates is increased relativeto the concentration of aptamer affinity complexes having faster, lessdesirable dissociation rates. In one embodiment, the slow off-rateenrichment process is a solution-based slow off-rate enrichment process.In this embodiment, a solution-based slow off-rate enrichment processtakes place in solution, such that neither the target nor the nucleicacids forming the aptamer affinity complexes in the mixture areimmobilized on a solid support during the slow off-rate enrichmentprocess. In various embodiments, the slow-off rate enrichment processcan include one or more steps, including the addition of and incubationwith a competitor molecule, dilution of the mixture, or a combination ofthese (e.g., dilution of the mixture in the presence of a competitormolecule). Because the effect of a slow off-rate enrichment processgenerally depends upon the differing dissociation rates of differentaptamer affinity complexes (i.e., aptamer affinity complexes formedbetween the target molecule and different nucleic acids in the candidatemixture), the duration of the slow off-rate enrichment process isselected so as to retain a high proportion of aptamer affinity complexeshaving slow dissociation rates while substantially reducing the numberof aptamer affinity complexes having fast dissociation rates. The slowoff-rate enrichment process may be used in one or more cycles during theSELEX process. When dilution and the addition of a competitor are usedin combination, they may be performed simultaneously or sequentially, inany order. The slow-off rate enrichment process can be used when thetotal target (protein) concentration in the mixture is low. In oneembodiment, when the slow off-rate enrichment process includes dilution,the mixture can be diluted as much as is practical, keeping in mind thatthe aptamer retained nucleic acids are recovered for subsequent roundsin the SELEX process. In one embodiment, the slow off-rate enrichmentprocess includes the use of a competitor as well as dilution, permittingthe mixture to be diluted less than might be necessary without the useof a competitor.

In one embodiment, the slow off-rate enrichment process includes theaddition of a competitor, and the competitor is a polyanion (e.g.,heparin or dextran sulfate (dextran)). Heparin and dextran have beenused in the identification of specific aptamers in prior SELEXselections. In such methods, however, heparin or dextran is presentduring the equilibration step in which the target and aptamer bind toform complexes. In such methods, as the concentration of heparin ordextran increases, the ratio of high affinity target/aptamer complexesto low affinity target/aptamer complexes increases. However, a highconcentration of heparin or dextran can reduce the number of highaffinity target/aptamer complexes at equilibrium due to competition fortarget binding between the nucleic acid and the competitor. By contrast,the presently described methods add the competitor after thetarget/aptamer complexes have been allowed to form, and therefore doesnot affect the number of complexes formed. Addition of competitor afterequilibrium binding has occurred between target and aptamer creates anon-equilibrium state that evolves in time to a new equilibrium withfewer target/aptamer complexes. Trapping target/aptamer complexes beforethe new equilibrium has been reached enriches the sample for slowoff-rate aptamers since fast off-rate complexes will dissociate first.

In another embodiment, a polyanionic competitor (e.g., dextran sulfateor another polyanionic material) is used in the slow off-rate enrichmentprocess to facilitate the identification of an aptamer that isrefractory to the presence of the polyanion. In this context,“polyanionic refractory aptamer” is an aptamer that is capable offorming an aptamer/target complex that is less likely to dissociate inthe solution that also contains the polyanionic refractory material thanan aptamer/target complex that includes a nonpolyanionic refractoryaptamer. In this manner, polyanionic refractory aptamers can be used inthe performance of analytical methods to detect the presence or amountor concentration of a target in a sample, where the detection methodincludes the use of the polyanionic material (e.g. dextran sulfate) towhich the aptamer is refractory.

Thus, in one embodiment, a method for producing a polyanionic refractoryaptamer is provided. In this embodiment, after contacting a candidatemixture of nucleic acids with the target, the target and the nucleicacids in the candidate mixture are allowed to come to equilibrium. Apolyanionic competitor is introduced and allowed to incubate in thesolution for a period of time sufficient to insure that most of the fastoff rate aptamers in the candidate mixture dissociate from the targetmolecule. Also, aptamers in the candidate mixture that may dissociate inthe presence of the polyanionic competitor will be released from thetarget molecule. The mixture is partitioned to isolate the highaffinity, slow off-rate aptamers that have remained in association withthe target molecule and to remove any uncomplexed materials from thesolution. The aptamer can then be released from the target molecule andisolated. The isolated aptamer can also be amplified and additionalrounds of selection applied to increase the overall performance of theselected aptamers. This process may also be used with a minimalincubation time if the selection of slow off-rate aptamers is not neededfor a specific application.

Thus, in one embodiment a modified SELEX process is provided for theidentification or production of aptamers having slow (long) off rateswherein the target molecule and candidate mixture are contacted andincubated together for a period of time sufficient for equilibriumbinding between the target molecule and nucleic acids contained in thecandidate mixture to occur. Following equilibrium binding an excess ofcompetitor molecule, e.g., polyanion competitor, is added to the mixtureand the mixture is incubated together with the excess of competitormolecule for a predetermined period of time. A significant proportion ofaptamers having off rates that are less than this predeterminedincubation period will dissociate from the target during thepredetermined incubation period. Re-association of these “fast” off rateaptamers with the target is minimized because of the excess ofcompetitor molecule which can non-specifically bind to the target andoccupy target binding sites. A significant proportion of aptamers havinglonger off rates will remain complexed to the target during thepredetermined incubation period. At the end of the incubation period,partitioning nucleic acid-target complexes from the remainder of themixture allows for the separation of a population of slow off-rateaptamers from those having fast off rates. A dissociation step can beused to dissociate the slow off-rate aptamers from their target andallows for isolation, identification, sequencing, synthesis andamplification of slow off-rate aptamers (either of individual aptamersor of a group of slow off-rate aptamers) that have high affinity andspecificity for the target molecule. As with conventional SELEX theaptamer sequences identified from one round of the modified SELEXprocess can be used in the synthesis of a new candidate mixture suchthat the steps of contacting, equilibrium binding, addition ofcompetitor molecule, incubation with competitor molecule andpartitioning of slow off-rate aptamers can be iterated/repeated as manytimes as desired.

The combination of allowing equilibrium binding of the candidate mixturewith the target prior to addition of competitor, followed by theaddition of an excess of competitor and incubation with the competitorfor a predetermined period of time allows for the selection of apopulation of aptamers having off rates that are much greater than thosepreviously achieved.

In order to achieve equilibrium binding, the candidate mixture may beincubated with the target for at least about 5 minutes, or at leastabout 15 minutes, about 30 minutes, about 45 minutes, about 1 hour,about 2 hours, about 3 hours, about 4 hours, about 5 hours or about 6hours.

The predetermined incubation period of competitor molecule with themixture of the candidate mixture and target molecule may be selected asdesired, taking account of the factors such as the nature of the targetand known off rates (if any) of known aptamers for the target.Predetermined incubation periods may be chosen from: at least about 5minutes, at least about 10 minutes, at least about 20 minutes, at leastabout 30 minutes, at least 45 about minutes, at least about 1 hour, atleast about 2 hours, at least about 3 hours, at least about 4 hours, atleast about 5 hours, at least about 6 hours.

In other embodiments a dilution is used as an off rate enhancementprocess and incubation of the diluted candidate mixture, targetmolecule/aptamer complex may be undertaken for a predetermined period oftime, which may be chosen from: at least about 5 minutes, at least about10 minutes, at least about 20 minutes, at least about 30 minutes, atleast about 45 minutes, at least about 1 hour, at least about 2 hours,at least about 3 hours, at least about 4 hours, at least about 5 hours,at least about 6 hours.

Embodiments of the present disclosure are concerned with theidentification, production, synthesis and use of slow off-rate aptamers.These are aptamers which have a rate of dissociation (t_(1/2)) from anon-covalent aptamer-target complex that is higher than that of aptamersnormally obtained by conventional SELEX. For a mixture containingnon-covalent complexes of aptamer and target, the t_(1/2) represents thetime taken for half of the aptamers to dissociate from theaptamer-target complexes. The t_(1/2) of slow dissociation rate aptamersaccording to the present disclosure is chosen from one of: greater thanor equal to about 30 minutes; between about 30 minutes and about 240minutes; between about 30 minutes to about 60 minutes; between about 60minutes to about 90 minutes, between about 90 minutes to about 120minutes; between about 120 minutes to about 150 minutes; between about150 minutes to about 180 minutes; between about 180 minutes to about 210minutes; between about 210 minutes to about 240 minutes.

A characterizing feature of an aptamer identified by a SELEX procedureis its high affinity for its target. An aptamer will have a dissociationconstant (k_(d)) for its target that is chosen from one of: less thanabout 1 μM, less than about 100 nM, less than about 10 nM, less thanabout 1 nM, less than about 100 pM, less than about 10 pM, less thanabout 1 pM.

Chemical Synthesis

Methods for the chemical synthesis of compounds provided in the presentdisclosure are described herein. These and/or other well-known methodsmay be modified and/or adapted in known ways in order to facilitate thesynthesis of additional compounds provided in the present disclosure.

With reference to Scheme 1, the present disclosure also provides amethod for the synthesis of a 3′-phosporamidite of a C-5 modifiedaminocarbonylpyrimidine. With reference to Scheme 1, it is worth notingthat the palladium catalyzed reaction of carbon monoxide and base withthe 5-substituted nucleoside is performed at carbon monoxide pressuresless than or equal to 2 atmospheres; more specifically from 0.1 to 2atmospheres (or 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1,1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2 atmospheres), and even morespecifically at 1 atmosphere. Reaction at these reduced pressures leadsto higher yields and purer product then previous methods which wereperformed at pressures between 3 and 4 atmospheres [50 psi].

With reference to Scheme 2, the present disclosure also provides amethod for the synthesis of a 5′-triphosphate of a C-5 modifiedaminocarbonylpyrimidine comprising: In certain embodiments theprotecting group is selected from the group consisting oftriphenylmethyl, p-anisyldiphenylmethyl, di-p-anisyldiphenylmethyl,p-dimethoxy trityltrityl, formyl, t-butyloxycarbonyl, benzyloxycarbonyl,2-chlorobenzyloxycarbonyl, 4-chlorobenzoyloxycarbonyl,2,4-dichlorobenzyloxycarbonyl, furfurylcarbonyl, t-amyloxycarbonyl,adamantyloxycarbonyl, 2-phenylpropyl-(2)-oxycarbonyl,2-(4-biphenyl)propyl-(2)-oxycarbonyl, 2-nitrophenylsulfenyl anddiphenylphosphinyl.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1: Synthesis of5-(N-benzylcarboxamide)-2′-deoxycytidine

This example provides the methods for making5-(N-benzylcarboxamide)-2′-deoxycytidine (or BndC; see Scheme 1 (4a)below). In brief, commercially-available 5-iodo-2′-deoxycytidine (3) wasconverted into the corresponding N-substituted-carboxamide (4a-c) bytreatment with the requisite aromatic primary amine RCH₂NH₂ (5-10 eq),carbon monoxide (</=1 atm), and (Ph₃P)₄Pd (2 mol %) inN,N-dimethylformamide (DMF) at room temperature for 24-48 hours (Scheme1). The excess primary amine and limited carbon monoxide were necessaryto limit formation of the 2-ketocarboxamide byproducts (see, e.g.,Uozumi, Y. et al. (2001) and Takacs et al., 2008). The modifiednucleoside products (4a-c) were readily purified by recrystallizationfrom alcohol.

The starting materials: 5-iodo-2′-deoxycytidine;5-iodo-2′-O-methyl-cytidine; 5-iodo-2′-deoxy-2′-flurocytidine werepurchased from ChemGenes Corporation (Wilmington, Mass. 01887, USA) orThermo Fisher Scientific Inc. (Waltham, Mass. 02454, USA). Carbonmonoxide (safety: poison gas) at 99.9% purity was purchased fromSpecialty Gases of America (Toledo, Ohio 43611, USA). All other reagentswere purchased from Sigma-Aldrich (Milwaukee, Wis. 53201, USA) and wereused as received.

5-(N-benzylcarboxamide)-2′-deoxycytidine (4a)

An argon-filled 1 L round-bottom flask was charged with:5-iodo-2′-deoxycytidine (30 g, 85 mmol); benzylamine (109.3 g, 1020mmol, 12 eq); and anhydrous N,N-dimethylformamide (DMF, 205 mL). Themixture was rapidly magnetically-stirred until all the solids haddissolved. The resulting solution was degassed by two cycles ofevacuation to 50 mm and refilling with argon. A mixture ofbis(dibenzylidineacetone)palladium(0) (978 mg, 1.7 mmol, 0.02 eq) andtriphenylphosphine (1.92 g, 7.3 mmol, 0.086 eq) was added and theresulting fine black suspension was rapidly stirred, evacuated to 50 mmand filled with carbon monoxide (1 atm) from a rubber balloon. Themixture was stirred at room temperature (˜20° C.) and periodicallyrefilled with carbon monoxide. After 26 hours, the reaction was found tobe complete by tlc analysis (silica gel, eluent: 15% methanol/85%dichloromethane (v/v), R_(f)(SM)=0.3, R_(f)(4a)=0.4). The reactionmixture was diluted with ethyl acetate (205 mL), filtered, and rinsedforward with 65% ethyl acetate/35% DMF (100 mL). The clear greenfiltrate was concentrated on a rotary evaporator (50-80° C., 1-2 mm)until all the solvents and most of the benzylamine had distilled. Thedark orange residue (˜75 g) was dissolved in hot abs. ethanol (650 mL)and rapidly hot-filtered to remove a small amount of insoluble flakes(˜2 g). The clear filtrate was allowed to cool with slow stirring andthe product crystallized as needles. After stirring overnight, theslurry was filtered and the cake washed with ice-cold ethanol (100 mL).After drying in vacuo, the product (4a) was obtained as a white,crystalline solid: 22.0 g, 72% yield. ¹H NMR (500 MHz, d6-DMSO): δ=8.73(t, J=5.8 Hz, 1H), 8.42 (s, 1H), 8.06 (bs, 1H), 7.75 (bs, 1H), 7.32 (m,4H), 7.25 (m, 1H), 6.14 (t, J=6.5 Hz, 1H), 5.24 (d, J=4.4 Hz, 1H), 5.03(t, J=5.5 Hz, 1H), 4.42 (dd, J=15.4, 7.2 Hz, 1H), 4.41 (dd, J=15.4, 7.3Hz, 1H), 4.26 (m, J=4.3 Hz, 1H), 3.83 (dd, J=7.9, 4.3 Hz, 1H), 3.64 (m,1H), 3.58 (m, 1H), 2.19 (m, 2H). ¹³C NMR (500 MHz, d6-DMSO): δ=165.88(1C), 163.97 (1C), 153.99 (1C), 144.26 (1C), 139.64 (1C), 128.80 (2C),127.60 (2C), 127.30 (1C), 99.20 (1C), 88.08 (1C), 86.29 (1C), 70.44(1C), 61.50 (1C), 42.72 (1C), 40.62 (1C). MS m/z: [M⁻] calcd forC₁₇H₁₉N₄O₅, 359.36; found, 359.1 (ESI⁻).

4-N-Acetyl-5-(N-benzylcarboxamide)-2′-deoxycytidine (5a)

A 1 L round bottom flask was charged with (4a) (20.0 g, 55.4 mmol), andanh. tetrahydrofuran (THF, 500 mL). The well-stirred mixture was treatedwith acetic anhydride (26.4 mL, 277 mmol, 5 eq) and the mixture washeated at 50° C. for 20 h. Tlc analysis of an aliquot (homogenized bydissolving in 50% methanol/50% dichloromethane) showed that the reactionwas complete (silica gel, eluent: 15% methanol/85% dichloromethane(v/v), R_(f)(4a)=0.4, R_(f)(5a)=0.6). The slurry was cooled to 5-10° C.for 1 hour, filtered, and washed with cold THF (40 mL). Drying in vacuoafforded the product (5a) as white, crystalline needles, 20.4 g, 91%yield. ¹H NMR (500 MHz, d6-DMSO): δ=11.35 (s, 1H), 8.98 (t, J=5.7 Hz,1H), 8.73 (s, 1H), 7.34 (d, J=4.4 Hz, 4H), 7.26 (m, J=4.3 Hz, 1H), 6.10(t, J=6.1 Hz, 1H), 5.28 (d, J=4.4 Hz, 1H), 5.09 (t, J=5.4 Hz, 1H), 4.44(dd, J=15.3, 8.1 Hz, 1H), 4.43 (dd, J=15.2, 8.1 Hz, 1H), 4.28 (dt,J=9.8, 4.0 Hz, 1H), 3.91 (dd, J=7.9, 4.0 Hz, 1H), 3.68 (m, 1H), 3.60 (m,1H), 2.41 (s, 3H), 2.34 (m, 1H), 2.22 (m, 1H). ¹³C NMR (500 MHz,d6-DMSO): δ=171.27 (1C), 165.49 (1C), 159.77 (1C), 153.19 (1C), 146.24(1C), 139.16 (1C), 128.82 (2C), 127.76 (2C), 127.41 (1C), 100.32 (1C),88.67 (1C), 87.50 (1C), 70.11 (1C), 61.17 (1C), 43.00 (1C), 40.78 (1C),26.70 (1C). MS m/z: [M⁻] calcd for C₁₉H₂₁N₄O₆, 401.40; found, 401.1(ESI⁻).

5′-O-(4,4′-Dimethoxytrityl)-4-N-acetyl-5-(N-benzylcarboxamide)-2′-deoxycytidine(6a)

A 250 mL round bottom flask was charged with (5a) (4.82 g, 12 mmol) andanhydrous pyridine (40 mL). The resulting colorless solution wasmagnetically-stirred as 4,4′-dimethoxytrityl chloride (4.47 g, 13.2mmol, 1.1 eq) was added in five portions over one hour. Theorange-yellow solution was stirred for 30 minutes more, quenched withethanol (4.2 mL, 72 mmol), and concentrated on the rotovap (1-2 mm, ≤35°C.). The resulting sticky orange residue (˜13 g) was partitioned withethyl acetate (100 mL) and cold, saturated aq. sodium bicarbonate (50mL). The organic layer was dried with sodium sulfate, filtered andconcentrated to leave a yellow foam. Purification by flashchromatography (silica gel, eluent: 1% triethylamine/99% ethyl acetate,R_(f)(6a)=0.4) afforded (6a) as a white foam, 6.1 g, 72% yield. ¹H NMR(500 MHz, d6-DMSO): δ=11.44 (s, 1H), 9.12 (t, J=5.5 Hz, 1H), 8.46 (s,1H), 7.38 (d, J=7.4 Hz, 2H), 7.25 (m, 12H), 6.84 (m, 4H), 6.10 (t, J=6.1Hz, 1H), 5.34 (d, J=4.7 Hz, 1H), 4.20 (m, 3H), 4.05 (m, 1H), 3.72 (d,J=1.7 Hz, 6H), 3.41 (dd, J=10.5, 6.0 Hz, 1H), 3.20 (dd, J=10.4, 3.5 Hz,1H), 2.44 (s, 3H), 2.39 (m, 1H), 2.25 (m, 1H). ¹³C NMR (500 MHz,d6-DMSO): δ=171.28 (1C), 165.38 (1C), 159.88 (1C), 158.53 (2C), 153.07(1C), 146.13 (1C), 145.26 (1C), 138.91 (1C), 136.00 (1C), 135.98 (1C),130.16 (2C), 130.11 (2C), 128.78 (2C), 128.28 (2C), 128.13 (2C), 127.84(2C), 127.46 (1C), 127.14 (1C), 113.60 (4C), 100.32 (1C), 88.04 (1C),86.86 (1C), 86.19 (1C), 70.69 (1C), 60.22 (1C), 55.43 (1C), 55.42 (1C),43.03 (1C), 40.70 (1C), 26.76 (1C). MS m/z: [M⁻] calcd for C₄₀H₃₉N₄O₈,703.77; found, 703.2 (ESI⁻).

5′-O-(4,4′-Dimethoxytrityl)-4-N-acetyl-5-(N-benzylcarboxamide)-2′-deoxycytidine-3′-O—(N,N-diisopropyl-O-2-cyanoethylphosphoramidite)(7a)

A 250 mL round bottom flask was charged with: (6a) (11.0 g, 15.6 mmol);anhydrous dichloromethane (40 mL);2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphordiamidite (5.9 mL, 18.7mmol, 1.2 eq); and finally, pyridine trifluoroacetate (3.61 g, 18.7mmol, 1.2 eq). After 30 min., tlc analysis showed that the reaction wascomplete (silica gel, 75% ethyl acetate/25% hexanes (v/v),R_(f)(6a)=0.2, R_(f)(7a)=0.7/0.8 [two isomers]). The entire reactionmixture was applied to a silica gel flash column preconditioned with 1%triethylamine/64% ethyl acetate/35% hexanes (until the eluent is basic),then eluted with 65% ethyl acetate/35% hexanes (argon-sparged). Theproduct-containing fractions were protected from air in sealed jarsunder argon and concentrated to afford (7a) as a colorless foam, 10.8 g,76% yield. ¹H NMR (500 MHz, d6-DMSO): δ=11.47 (s, 1H), 9.11 (bs, 1H),8.57/8.54 (s, 1H), 7.37 (m, 2H), 7.24 (m, 12H), 6.84 (m, 4H), 6.10 (m,1H), 4.40 (m, 1H), 4.21 (m, 3H), 3.70 (m, 8H), 3.55 (m, 2H), 3.28 (m,2H), 2.75 (t, J=5.9 Hz, 1H), 2.64 (t, J=5.9 Hz, 1H), 2.55 (m, 1H), 2.42(m, 4H), 1.11 (m, 9H), 0.98 (d, J=6.8 Hz, 3H). ³¹P NMR (500 MHz,d6-DMSO): δ=147.55/147.37 (s, 1P). MS m/z: [M⁻] calcd for C₄₅H₅₆N₆O₉P,903.99; found, 903.3 (ESI⁻).

For preparation of 2-cyanoethylphosphoramidite reagents (CEP reagents),the 5-N-benyzlarboxame (4a-c) were selectively N-protected by stirringwith acetic anhydride (no base) in tetrahydrofuran (THF), and then5′-O-protected as the (4,4′-dimethoxytrityl)-derivatives (6a-c) byreaction with 4,4′-dimethoxytrityl chloride in pyridine (see, e.g., Rosset al., 2006). Synthesis of the high purity (>98.0%) CEP reagents (7a-c)was completed by pyridinium trifluoroacetate-catalyzed condensation ofthe 3′-alcohol with 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphoramidite (see, e.g., Sanghvi, et al., 2000) and finalpurification by silica gel flash chromatography with degassed solvents(see, e.g., Still et al., 1978).

For preparation of 5′-triphosphate reagents (TPP reagent; Scheme 2), the5′-O-DMT-protected nucleosides (6a-c) were peracetylated with aceticanhydride in pyridine, followed by cleavage of the DMT and 4-N-acetylprotecting groups with 1,1,1,3,3,3-hexafluoro-2-propanol (see, e.g.,Leonard and Neelima, 1995). The resulting crystalline 3′-O-acetatenucleosides (8a-c) were converted into the crude 5′-O-triphosphates bythe Ludwig-Eckstein process. These chemically modified nucleotidesgenerally require a two-stage purification process: anion-exchangechromatography (AEX), followed by reverse phase preparative-HPLC inorder to obtain high purity (>%90).

Ludwig, J. and Eckstein, F. (1989) Rapid and Efficient Synthesis ofNucleoside 5′-O-(1-Thiotriphosphates), 5′-Triphosphates and2′,3′-Cyclophosphorothioates Using2-Chloro-4H-1,3,2-benzodioxaphophorin-4-one. J. Org. Chem., 54, 631-635,which is hereby incorporated by reference in its entirety.

The carboxyamidation reaction was also suitable for preparation ofnuclease-resistant ribo-sugar analogues (see, e.g., Ito et al., 2003)(Scheme 4), for example, 5-(N-benzylcarboxamide)-2′-O-methyl-cytidine(12) and -5-(N-3-phenylpropylcarboxamide)-2′-deoxy-2′-fluoro-cytidine(13).

5-(N-Benzylcarboxamide)-2′-O-methyl-cytidine (12)

Prepared from 5-iodo-2′-O-methylcytidine as described for (4a), exceptthat the product was crystallized from hot 2-propanol (12 mL/g)affording (12) as a felty white solid, 79% yield. ¹H NMR (500 MHz,d6-DMSO): δ=8.57 (t, J=5.9 Hz, 1H), 8.57 (s, 1H), 8.05 (bs, 1H), 7.85(bs, 1H), 7.33 (m, 4H), 7.25 (m, 1H), 5.85 (d, J=3.1 Hz, 1H), 5.27 (t,J=5.4 Hz, 1H), 5.11 (d, J=6.8 Hz, 1H), 4.43 (dd, J=15.4, 10.4 Hz, 1H),4.40 (dd, J=15.3, 10.4 Hz, 1H), 4.17 (dd, J=6.7, 5.2 Hz, 1H), 3.87 (dt,J=6.8, 3.2 Hz, 1H), 3.80 (dd, J=5.0, 3.2 Hz, 1H), 3.77 (m, 1H), 3.61(ddd, J=12.2, 5.3, 3.4 Hz, 1H), 3.44 (s, 3H). ¹³C NMR (500 MHz,d6-DMSO): δ=165.43 (1C), 163.57 (1C), 153.49 (1C), 143.99 (1C), 139.16(1C), 128.40 (2C), 127.22 (2C), 126.90 (1C), 98.97 (1C), 87.95 (1C),84.28 (1C), 83.05 (1C), 67.67 (1C), 59.92 (1C), 57.70 (1C), 42.40 (1C).MS m/z: [M⁻] calcd for C₁₈H₂₁N₄O₆, 389.39; found, 389.1 (ESI⁻).

5-(N-3-Phenylpropyl)-2′-deoxy-2′-fluoro-cytidine (13)

Prepared from 5-iodo-2′-deoxy-2′-fluoro-cytidine as described for (4c)as felty white solid (53% yield). ¹H NMR (500 MHz, d6-DMSO): δ=8.52 (s,1H), 8.07 (bs, 1H), 7.95 (t, J=5.4 Hz, 1H), 7.85 (bs, 1H), 7.22 (t,J=7.4, 5H), 5.91 (d, J=17.6 Hz, 1H), 5.58 (d, J=6.6 Hz, 1H), 5.32 (t,J=5.3 Hz, 1H), 4.99 (dd, J=53.2, 3.9 Hz, 1H), 4.27 (m, 1H), 3.92 (d,J=8.3 Hz, 1H), 3.86 (m, 1H), 3.58 (ddd, J=12.5, 5.4, 2.9 Hz, 1H), 3.19(dd, J=12.7, 5.3 Hz, 2H), 2.61 (t, J=7.5 Hz, 2H), 1.78 (m, J=7.3 Hz,2H). ¹³C NMR (500 MHz, d6-DMSO): δ=165.22 (s, 1C), 163.74 (s, 1C),153.34 (s, 1C), 143.82 (s, 1C), 141.73 (s, 1C), 128.42 (s, 2C), 128.35(s, 2C), 125.81 (s, 1C), 99.26 (1C), 94.02 (d, J=736.6 Hz, 1C), 88.65(d, J=134.7 Hz, 1C), 83.07 (s, 1C), 67.00 (d, J=65 Hz, 1C), 59.24 (s,1C), 38.65 (s, 1C), 32.64 (s, 1C), 30.73 (s, 1C). ¹⁹F NMR (400 MHz,d6-DMSO): δ=−200.82 (ddd, J=19.0, 6.2, 56.6 Hz, 1F). MS m/z: [M⁻] calcdfor C₁₉H₂₂N₄O₅, 405.41; found, 405.1 (ESI⁻).

The Ludwig-Eckstein process was used to convert the3′-O-acetyl-protected intermediates (8a-c) into crude 5′-O-triphosphates(9a-c). An alternative two-stage preparative HPLC purification was usedfor these chemically-modified nucleotides.

3′-O-Acetyl-5-(N-1-benzylcarboxamide)-2′-deoxycytidine (8a)

An argon purged 50 mL round bottom flask was charged with (6a) (900 mg,1.35 mmol), anh. pyridine (9 mL) and acetic anhydride (0.63 mL, 6.75mmol). After 18 h at room temperature, the solvent was evaporated invacuo (1 mm, 30° C.) to yield a tan foam which was dissolved in1,1,1,3,3,3-hexafluoro-2-propanol (9 mL) and heated at 50° C. underargon. After 6 h, the reaction mixture was poured into a rapidlystirring mixture of methanol (15 mL) and toluene (10 mL). The resultingorange solution was concentrated (1 mm, 30° C.) to yield a red oilyresidue which, upon mixing with ethyl acetate (6 mL), gave a crystallineslurry. Crystallization was further enhanced with the addition ofhexanes (1 mL). The mixture was stirred overnight and filtered, washingthe filter cake with 50:50 ethyl acetate:hexanes. The product (8a) wasisolated, after drying, as a pale gray solid (405 mg), 75% yield: ¹H NMR(500 MHz, d6-DMSO): δ=8.82 (t, J=5.8 Hz, 1H), 8.41 (s, 1H), 8.09 (bs,1H), 7.81 (bs, 1H), 7.33 (m, 4H), 7.25 (m, 1H), 6.17 (dd, J=8.0, 6.0 Hz,1H), 5.24 (dt, J=6.2, 1.8 Hz, 1H), 5.13 (t, J=5.6 Hz, 1H), 4.43 (dd,J=15.4, 13.3 Hz, 1H), 4.19 (dd, J=15.3, 13.2 Hz, 1H), 4.06 (dd, J=5.9,3.7 Hz, 1H), 3.65 (m, 2H), 2.45 (m, 1H), 2.34 (ddd, J=14.2, 5.9, 1.5 Hz,1H), 2.07 (s, 3H). ¹³C NMR (500 MHz, d6-DMSO): δ=170.05 (1C), 165.34(1C), 163.54 (1C), 153.44 (1C), 143.94 (1C), 139.20 (1C), 128.34 (2C),127.16 (2C), 126.85 (1C), 99.06 (1C), 85.96 (1C), 85.06 (1C), 74.63(1C), 61.18 (1C), 42.28 (1C), 37.31 (1C), 20.85 (1C). MS m/z: [M⁻] calcdfor C₁₉H₂₁N₄O₆, 401.40; found, 401.1 [M]⁻.

3′-O-Acetyl-5-(N-1-naphthylmethylcarboxamide)-2′-deoxycytidine (8b)

Prepared from (6b), as described for (8a), as a pale gray solid, 54%yield: ¹H NMR (500 MHz, d6-DMSO): δ=8.89 (t, J=5.8 Hz, 1H), 8.44 (s,1H), 8.14 (d, J=8.4 Hz, 1H), 8.11 (bs, 1H), 7.96 (dd, J=8.6, 1.3 Hz,1H), 7.86 (dd, J=6.6, 2.8 Hz, 1H), 7.84 (bs, 1H), 7.57 (m, 2H), 7.49 (m,2H), 6.15 (dd, J=8.2, 6.0 Hz, 1H), 5.23 (dt, J=6.2, 1.9 Hz, 1H), 5.13(t, J=5.8 Hz, 1H), 4.94 (dd, J=15.5, 5.8 Hz, 1H), 4.86 (dd, J=15.7, 5.4Hz, 1H), 4.05 (dt, J=8.1, 1.8 Hz, 1H), 3.62 (m, 2H), 2.45 (m, 1H), 2.33(ddd, J=12.6, 6.1, 1.8 Hz, 1H), 2.06 (s, 3H). ¹³C NMR (500 MHz,d6-DMSO): δ=170.51 (1C), 165.78 (1C), 164.02 (1C), 153.90 (1C), 144.52(1C), 134.57 (1C), 133.74 (1C), 131.27 (1C), 129.02 (1C), 128.03 (1C),126.80 (1C), 126.31 (1C), 125.94 (1C), 125.54 (1C), 123.78 (1C), 99.52(1C), 86.60 (1C), 85.55 (1C), 70.12 (1C), 61.67 (1C), 40.80 (1C), 37.75(1C), 21.32 (1C). MS m/z: [M⁻] calcd for C₂₃H₂₃N₄O₆, 451.46; found,451.1 (ESI⁻).

3′-O-Acetyl-5-(N-3-phenylpropylcarboxamide)-2′-deoxycytidine (8c)

Prepared from (6c), as described for (8a), as a white solid, 74% yield:¹H NMR (500 MHz, d6-DMSO): δ=8.37 (s, 1H), 8.26 (t, J=5.3 Hz, 1H), 8.06(bs, 1H), 7.79 (bs, 1H), 7.23 (m, 5H), 6.16 (dd, J=7.9, 6.1 Hz, 1H),5.24 (dt, J=4.2, 2.1 Hz, 1H), 5.18 (t, J=5.6 Hz, 1H), 4.06 (dd, J=5.7,3.6 Hz, 1H), 3.65 (m, 2H), 3.19 (dd, J=12.8, 6.2 Hz, 2H), 2.62 (t, J=7.5Hz, 2H), 2.43 (m, 1H), 2.34 (m, 1H), 2.06 (s, 3H), 1.78 (m, J=7.4 Hz,2H). ¹³C NMR (500 MHz, d6-DMSO): δ=170.52 (1C), 165.68 (1C), 164.00(1C), 153.94 (1C), 144.09 (1C), 142.13 (1C), 128.80 (2C), 127.75 (2C),126.21 (1C), 99.80 (1C), 86.42 (1C), 85.06 (1C), 75.07 (1C), 61.63 (1C),39.12 (1C), 37.91 (1C), 33.04 (1C), 31.18 (1C), 21.31 (1C). MS m/z: [M⁻]calcd for C₂₁H₂₅N₄O₆, 429.45; found, 429.1 (ESI⁻).

Example 2: Synthesis of5-(N-1-Napthylmethyl)-2′-deoxycytidine-5-carboxamide

This example provides the methods for making5-(N-1-Napthylmethyl)-2′-deoxycytidine-5-carboxamide (or NapdC; seeScheme 1 (4b) in Example 1).

The starting materials: 5-iodo-2′-deoxycytidine;5-iodo-2′-O-methyl-cytidine; 5-iodo-2′-deoxy-2′-flurocytidine werepurchased from ChemGenes Corporation (Wilmington, Mass. 01887, USA) orThermo Fisher Scientific Inc. (Waltham, Mass. 02454, USA). Carbonmonoxide (safety: poison gas) at 99.9% purity was purchased fromSpecialty Gases of America (Toledo, Ohio 43611, USA). All other reagentswere purchased from Sigma-Aldrich (Milwaukee, Wis. 53201, USA) and wereused as received.

5-(N-1-Napthylmethyl)-2′-deoxycytidine-5-carboxamide (4b)

Prepared as described for (4a), using 1-naphthylmethylamine (6 eq) inplace of benzylamine, with a reaction time of 48 hours at roomtemperature. After concentrating the reaction mixture, the residue wasextracted with diisopropyl ether (˜40 mL/g) to remove most of the excess1-naphthylmethylamine. The residue was crystallized from hot methanol(50 mL/g), with hot filtration, to afford the product (4b; Scheme 1;Example 1) as a white solid, 40% yield. ¹H NMR (500 MHz, d6-DMSO):δ=8.81 (t, J=5.5 Hz, 1H), 8.43 (s, 1H), 8.14 (d, J=4.4, 1H), 8.09 (bs,1H), 7.96 (m, 1H), 7.79 (m, 1H), 7.75 (bs, 1H), 7.53 (m, 4H), 6.14 (t,J=6.6 Hz, 1H), 5.24 (d, J=4.3 Hz, 1H), 5.01 (t, J=5.6 Hz, 1H), 4.90 (dd,J=15.6, 13.4 Hz, 1H), 4.89 (dd, J=15.5, 13.2 Hz, 1H), 4.26 (m, J=4.1 Hz,1H), 3.84 (dd, J=8.4, 4.4 Hz, 1H), 3.58 (m, 2H), 2.20 (m, 2H). ¹³C NMR(500 MHz, d6-DMSO): δ=165.45 (1C), 163.58 (1C), 153.57 (1C), 143.93(1C), 136.07 (1C), 134.20 (1C), 133.32 (1C), 128.61 (1C), 127.59 (1C),126.34 (1C), 125.89 (1C), 125.53 (1C), 125.07 (1C), 123.36 (1C), 98.82(1C), 87.71 (1C), 85.99 (1C), 70.13 (1C), 61.16 (1C), 42.42 (1C), 40.14(1C). MS m/z: [M⁻] calcd for C₂₁H₂₁N₄O₅, 409.42; found, 409.1 (ESI⁻).

4-N-Acetyl-5-(N-1-naphthylmethylcarboxamide)-2′-deoxycytidine (5b)

A 100 mL round bottom flask was charged with (4b) (1.17 g, 2.85 mmol)and anh. tetrahydrofuran (26 mL) and stirred to form a gray-whiteslurry. Acetic anhydride (1.4 mL, 14.3 mmol, 5 eq) was added to themixture dropwise while stirring at room temperature. The reactionmixture was stirred and heated to 50° C. for 21 h. An aliquot was pulledfor TLC analysis (silica gel, eluent: 10% methanol/90% dichloromethane(v/v), R_(f)(4b)=0.61, R_(f)(5b)=0.12) which indicated that the reactionwas complete. The reaction flask was transferred to an ice bath andstirred for >1 h. The mixture was then filtered and the filter cake wasrinsed with chilled isopropyl ether. The resulting solids were collectedand further evaporated in vacuo to yield fine gray-white crystals (1.01g, 78.2% yield). ¹H NMR (500 MHz, d6-DMSO): δ=11.35 (s, 1H), 9.07 (t,J=4.6 Hz, 1H), 8.74 (s, 1H), 8.15 (d, J=8.7 Hz, 1H), 7.96 (m, 1H), 7.87(m, 1H), 7.53 (m, 4H), 6.11 (t, J=6.2 Hz, 1H), 5.29 (d, J=4.2 Hz, 1H),5.08 (t, J=5.4 Hz, 1H), 4.92 (dd, J=15.5, 10.1 Hz, 1H), 4.91 (dd,J=15.7, 9.7 Hz, 1H), 4.28 (dt, J=9.4, 3.8 Hz, 1H), 3.92 (dd, J=7.6, 3.9Hz, 1H), 3.64 (m, 1H), 3.58 (m, 1H), 2.42 (s, 3H), 2.35 (m, 1H), 2.22(m, 1H). ¹³C NMR (500 MHz, d6-DMSO): δ=171.27 (1C), 165.53 (1C), 159.77(1C), 153.20 (1C), 146.30 (1C), 136.48 (1C), 134.17 (1C), 133.74 (1C),131.26 (1C), 129.02 (1C), 128.12 (1C), 126.83 (1C), 126.33 (1C), 125.95(1C), 123.80 (1C), 100.38 (1C), 88.74 (1C), 87.63 (1C), 70.25 (1C),61.29 (1C), 41.13 (1C), 40.92 (1C), 26.71 (1C). MS m/z: [M⁻] calcd forC₂₃H₂₃N₄O₆, 451.46; found, 451.1 (ESI⁻).

5′-O-(4,4′-Dimethoxytrityl)-4-N-Acetyl-5-(N-1-naphthylmethylcarboxamide)-2′-deoxycytidine(6b)

Prepared as described for 6b) as a colorless solid in 59% yield. ¹H NMR(500 MHz, d6-DMSO): δ=11.40 (s, 1H), 9.35 (bt, 1H), 8.48 (s, 1H), 8.02(m, 1H), 7.96 (m, 1H), 7.86 (d, J=8.4 Hz, 1H), 7.54 (m, 2H), 7.40 (m,2H), 7.35 (m, 1H), 7.25 (m, 8H), 6.85 (d, J=8.9 Hz, 4H), 6.09 (t, J=6.1Hz, 1H), 5.32 (d, J=3.7 Hz, 1H), 4.72 (dd, J=14.9, 4.8 Hz, 1H), 4.60(dd, J=15.1, 3.4 Hz, 1H), 4.16 (dt, J=10.9, 4.7 Hz, 1H), 4.04 (m, 1H),3.70 (s, 6H), 3.29 (dd, J=10.5, 6.4 Hz, 1H), 3.18 (dd, J=10.4, 7.0 Hz,1H), 2.43 (s, 3H), 2.38 (m, 1H), 2.26 (m, 1H). ¹³C NMR (500 MHz,d6-DMSO): δ=171.25 (1C), 165.37 (1C), 159.88 (1C), 158.52 (1C), 158.54(1C), 153.03 (1C), 146.32 (1C), 145.31 (1C), 136.05 (1C), 135.97 (1C),133.92 (1C), 133.74 (1C), 131.22 (1C), 130.20 (2C), 130.11 (2C), 129.05(1C), 128.30 (2C), 128.15 (2C), 127.16 (1C), 126.81 (1C), 126.33 (1C),125.85 (1C), 125.80 (1C), 123.65 (1C), 113.61 (4C), 100.49 (1C), 88.12(1C), 86.79 (1C), 86.17 (1C), 70.59 (1C), 64.40 (1C), 55.41 (2C), 41.01(1C), 40.70 (1C), 40.82 (1C), 26.76 (1C). MS m/z: [M⁻] calcd forC₄₄H₄₁N₄O₈, 753.83; found, 753.21 (ESI⁻).

5′-O-(4,4′-Dimethoxytrityl)-4-N-Acetyl-5-(N-1-naphthylmethylcarboxamide)-2′-deoxycytidine-3′-O—(N,N-diisopropyl-O-2-cyanoethylphosphoramidite)(7b)

Prepared as described for (7a) as a white foam (88% yield). ¹H NMR (500MHz, d6-DMSO): δ=11.41 (s, 1H), 9.13 (bs, 1H), 8.56/8.54 (s, 1H), 8.01(m, 1H), 7.95 (m, 1H), 7.85 (m, 1H), 7.53 (m, 2H), 7.37 (m, 2H), 7.24(m, 9H), 6.83 (m, 4H), 6.06 (m, 1H), 4.66 (m, 2H), 4.39 (m, 1H), 4.16(m, 1H), 3.68 (m, 8H), 3.52 (m, 2H), 3.28 (m, 1H), 3.20 (m, 1H), 2.74(t, J=5.8 Hz, 1H), 2.63 (t, J=5.9 Hz, 1H), 2.45 (m, 5H), 1.09 (m, 9H),0.96 (d, J=6.8 Hz, 3H). ³¹P NMR (500 MHz, d6-DMSO): δ=146.93/146.69 (s,1P). MS m/z: [M⁻] calcd for C₅₃H₅₈N₆O₉P, 954.05; found, 953.3 (ESI⁻).

Example 3: Synthesis of 5-(N-3-phenylpropylcarboxamide)-2′-deoxycytidine

This example provides the methods for making5-(N-3-phenylpropylcarboxamide)-2′-deoxycytidine (or PPdC; see Scheme 1(4c) in Example 1).

The starting materials: 5-iodo-2′-deoxycytidine;5-iodo-2′-O-methyl-cytidine; 5-iodo-2′-deoxy-2′-flurocytidine werepurchased from ChemGenes Corporation (Wilmington, Mass. 01887, USA) orThermo Fisher Scientific Inc. (Waltham, Mass. 02454, USA). Carbonmonoxide (safety: poison gas) at 99.9% purity was purchased fromSpecialty Gases of America (Toledo, Ohio 43611, USA). All other reagentswere purchased from Sigma-Aldrich (Milwaukee, Wis. 53201, USA) and wereused as received.

5-(N-3-phenylpropylcarboxamide)-2′-deoxycytidine (4c)

Prepared as described for (4a) (40 nmol scale), using3-phenylpropylamine (6 eq) in place of benzylamine and a reaction timeof 48 hours at room temperature. After removal of the solvents on therotovap, the residue was triturated with diethyl ether (˜30 mL/g) toextract the excess 3-phenylpropylamine

and the gummy residue was dissolved in hot ethanol, stirred at roomtemperature for 18 h, followed by stirring at 0° C. for 1 h. Theresulting mixture was filtered and the mother liquor was evaporatedresulting in a brown resin. This was dissolved in warm mixture ofdichloromethane and water. After standing and stirring at roomtemperature, white feathery crystals formed in the organic layer, and inthe aqueous layer as well. The triphasic mixture was filtered and thefilter cake was washed with diethyl ether to afford (4c) as a fluffywhite solid (10.78 g, 69.5% yield). ¹H NMR (500 MHz, d6-DMSO): δ=8.39(s, 1H), 8.13 (t, J=5.3 Hz, 1H), 8.05 (bs, 1H), 7.71 (bs, 1H), 7.28 (t,J=7.4, 2H), 7.22 (d, J=7.0, 2H), 7.17 (t, J=7.4, 1H), 6.13 (t, J=6.4 Hz,1H), 5.22 (d, J=4.3 Hz, 1H), 5.07 (t, J=5.5 Hz, 1H), 4.26 (dt, J=9.4,4.1 Hz, 1H), 3.83 (dd, J=7.8, 3.9 Hz, 1H), 3.66 (m, 1H), 3.58 (m, 1H),3.19 (dd, J=12.9, 6.7 Hz, 2H), 2.61 (t, J=7.5 Hz, 2H), 2.19 (m, 2H),1.78 (m, J=7.4 Hz, 2H). ¹³C NMR (500 MHz, d6-DMSO): δ=165.34 (1C),163.56 (1C), 153.60 (1C), 143.53 (1C), 141.70 (1C), 128.38 (2C), 128.33(2C), 125.78 (1C), 98.99 (1C), 87.63 (1C), 85.86 (1C), 69.82 (1C), 60.96(1C), 40.36 (1C), 38.58 (1C), 32.63 (1C), 32.63 (1C). MS m/z: [M⁻] calcdfor C₁₉H₂₃N₄O₅, 387.42; found, 387.1 (ESI⁻).

4-N-Acetyl-5-(N-3-phenylpropyl)carboxamide-2′-deoxycytidine (5c)

A solution of (4c) (10.8 g, 28 mmol) in anh.THF (100 mL) was stirred andtreated dropwise with acetic anhydride (3 eq). The solution was stirredfor 18 hours at room temperature affording a thin suspension. Themixture was slowly diluted by drop wise addition of diisopropyl ether(35 mL). The solids were isolated by filtration and dried in vacuo toafford (5c) as a white solid (8.44 g, 70.5% yield). ¹H NMR (400 MHz,d6-DMSO): δ=11.34 (s, 1H), 8.69 (s, 1H), 8.41 (t, J=5.2 Hz, 1H), 7.23(m, 5H), 6.09 (t, J=6.0 Hz, 1H), 5.15 (bs, 2H), 4.27 (m, 1H), 3.90 (dd,J=9.6, 3.8 Hz, 1H), 3.68 (m, 1H), 3.59 (m, 1H), 3.21 (dd, J=12.3, 7.0 Hz2H), 2.62 (m, 2H), 2.40 (s, 3H), 2.33 (m, 1H), 2.21 (m, 1H), 1.79 (m,2H). ¹³C NMR (400 MHz, d6-DMSO): δ=171.21 (C), 165.34 (1C), 159.73 (1C),153.21 (1C), 146.01 (1C), 142.08 (1C), 128.80 (2C), 128.75 (2C), 126.22(1C), 99.14 (1C), 88.61 (1C), 87.41 (1C), 69.88 (1C), 61.05 (1C), 41.04(1C), 39.22 (1C), 33.01 (1C), 30.97 (1C), 26.67 (1C). MS m/z: [M⁻] calcdfor C₂₁H₂₅N₄O₆, 429.45; found, 429.1 (ESI⁻).

5′-O-(4,4′-Dimethoxytrityl)-4-N-acetyl-5-(N-3-phenylpropyl)carboxamide-2′-deoxycytidine(6c)

Prepared from (5c; Scheme 1; Example 1), as described for (6a) inExample 1 as a white foam (64.6% yield). ¹H NMR (500 MHz, d6-DMSO):δ=11.38 (s, 1H), 8.56 (t, J=5.2 Hz, 1H), 8.37 (s, 1H), 7.35 (d, J=7.4Hz, 2H), 7.21 (m, 12H), 6.80 (m, 4H), 6.11 (t, J=6.0 Hz, 1H), 5.32 (d,J=4.8 Hz, 1H), 4.16 (dt, J=10.8, 4.7 Hz, 1H), 4.04 (m, 1H), 3.70 (d,J=2.2 Hz, 6H), 3.26 (dd, J=10.6, 6.1 Hz, 1H), 3.21 (dd, J=10.5, 3.3 Hz,1H), 3.03 (m, 1H), 2.95 (m, 1H), 2.49 (s, 2H), 2.43 (s, 3H), 2.39 (m,1H), 2.23 (m, 1H), 1.57 (m, 2H). ¹³C NMR (500 MHz, d6-DMSO): δ=170.77(1C), 164.81 (1C), 159.41 (1C), 158.07 (1C), 158.06 (1C), 152.69 (1C),145.18 (1C), 144.81 (1C), 141.53 (1C), 135.51 (1C), 135.50 (1C), 129.70(2C), 129.61 (2C), 128.32 (2C), 128.29 (2C), 127.81 (2C), 127.66 (2C),126.66 (1C), 125.79 (1C), 113.13 (4C), 100.37 (1C), 87.47 (1C), 86.40(1C), 85.72 (1C), 70.03 (1C), 59.80 (1C), 54.99 (1C), 54.97 (1C), 40.48(1C), 38.92 (1C), 32.64 (1C) 32.29 (1C), 26.27 (1C). MS m/z: [M⁻] calcdfor C₄₂H₄₃N₄O₈, 731.83; found, 731.2 (ESI⁻).

5′-O-(4,4′-Dimethoxytrityl)-4-N-acetyl-5-(N-3-phenylpropylcarboxamide)-2′-deoxycytidine-3′-O—(N,N-diisopropyl-O-2-cyanoethylphosphoramidite)(7c)

Prepared from (6c; Scheme 1; Example 1), as described for (7a) inExample 1. A 500 mL round bottom flask containing 6c (7.11 g, 9.70 mmol)under an argon atmosphere was charged with anh. dichloromethane (97 mL).Anhydrous N, N-diisopropylethylamine (3.4 mL, 19.4 mmol) was added tothe flask, and mixture was chilled to 0° C. while stirring. Over thecourse of a half hour, 2-cyanoethyldiisopropyl chlorophosphoramidite(2.6 mL, 11.6 mmol) was added dropwise to the rapidly stirring mixture.The mixture was allowed to slowly warm to room temperature while itstirred. After 17 hour, the reaction was sampled for TLC, which showedthat the reaction was complete (silica gel; eluent 75% ethyl acetate/25%hexanes (v/v), R_(f)(6c)=0.10, R_(f)(7b)=0.46/0.56 [two isomers]). Thereaction mixture was transferred to a 250 mL separatory funnel usingtoluene and quenched by washing with cold, argon sparged 2% sodiumbicarbonate solution (2×, 400 mL/wash). The organic layer was collectedand evaporated until the majority of the dichloromethane had beenremoved. The organic layer was returned to the separatory funnel withchilled argon-sparged toluene and washed with chilled argon-spargeddeionized water (2×, 400 mL/wash). The organic layer was then dilutedwith chilled argon-sparged ethyl acetate and washed with brine (1×, 400mL). The organic layer was collected, dried over sodium sulfate,filtered and evaporated. The worked-up reaction mixture was dissolvedwith dichloromethane and loaded onto a pre-conditioned column (preparedas for (6b)) and eluted with chilled, argon sparged mobile phase (80%ethyl acetate/20% hexanes) and product containing fractions werecollected in sealed argon-purged bottles to limit product contact withair. Product containing fractions were evaporated at <40° C. to yield awhite foam (6.16 g, 68.0% yield). ¹H NMR (500 MHz, d6-DMSO): δ=11.40 (s,1H), 8.56 (m, 1H), 8.47/8.43 (s, 1H), 7.35 (m, 2H), 7.20 (m, 12H), 6.80(m, 4H), 6.08 (m, 1H), 4.40 (m, 1H), 4.18 (m, 1H), 3.70 (m, 8H), 3.53(m, 2H), 3.30 (m, 2H), 3.00 (m, 2H), 2.75 (t, J=5.9 Hz, 1H), 2.63 (t,J=5.9 Hz, 1H), 2.56 (m, 1H), 2.50 (m, 2H), 2.40 (m, 4H), 1.59 (m, 2H),1.11 (m, 9H), 0.97 (d, J=6.8 Hz, 3H). ³¹P NMR (500 MHz, d6-DMSO):δ=147.60/147.43 (s, 1P) MS m/z: [M⁻] calcd for C₅₁H₆₀N₆O₉P, 932.05;found, 931.4 (ESI.

Example 4: Nucleoside Triphosphate Purification by Two-Stage PreparativeHPLC

This example provides the methods for the purification of nucleosidetriphosphates.

The crude triphosphates (9a-c) were purified via two orthogonalpreparative HPLC techniques: anion exchange chromatography to separatethe nucleoside triphosphate from other nucleoside by-products (such asdiphosphate and monophosphate) and reversed-phase chromatography toremove residual by-products of reaction reagents.

Anion exchange chromatography was performed in two injections for each0.5 mmol reaction using an HPLC column packed with Source 15Q resin,eluting with a linear elution gradient of two triethylammoniumbicarbonate buffers (Table 1). The desired triphosphate was usually thefinal material to elute from the column, as a broad peak with 10-12minutes width on the HPLC chromatogram. Fractions were analyzed andproduct-containing fractions were combined and evaporated in a GenevacVC 3000D evaporator to produce a colorless to light tan resin. This wasreconstituted in deionized water and applied in a single injection forreversed phase purification on a Novapak HRC18 prep column eluting witha linear gradient of acetonitrile in triethylammonium acetate buffer(Table 2). Fractions containing pure triphosphate were combined andevaporated to produce a colorless to light tan resin.

Final pure triphosphates (9a-c) were reconstituted in deionized waterfor analysis and quantitated using a Hewlett Packard 8452A Diode ArraySpectrophotometer at 240 nm (Table 3).

TABLE 1 AEX Purification Conditions HPLC system Waters 625HPLC/486detector @ 240 or 278 nm Column Source 15Q 196 mL (GE Healthcare PN:17-1947-05) Mobile Phase A: 10 mM Triethylammonium bicarbonate/10%Acetonitrile, B: 1M Triethylammonium bicarbonate/10% AcetonitrileGradient (% Buffer B) 5%-70% Run Time; flow rate; 50 min; 35 mL/min; 50mL fraction size Analytical Column Dionex DNA-Pac PA100 column (ThermoScientific, PN: 043010)

TABLE 2 RP-HPLC Purification Conditions HPLC system Waters 625HPLC/486detector @ 240 nm Column Waters Novapak HRC18, 19 mm × 300 mm (PNWAT025822) Mobile Phase A: 100 mM Triethylammonium acetate B: 100%Acetonitrile Gradient (% Buffer B) 0%-50% Run Time; flow rate; 30 min;12 mL/min; 25 mL fraction size Analytical Column Waters Symmetry column(PN: WAT054215)

TABLE 3 Triphosphate Yields and Purities Extinction Purity: Purity:Triphos- Coefficient Yield Yield Analytical Analytical phate (est3b)μmoles Percent AEX RP-HPLC (9a) 13,700 cm⁻¹ M⁻¹ 43  9% no data 92.6%(9b) 20,000 cm⁻¹ M⁻¹ 121 24% 95.5% 98.2% (9c) 13,700 cm⁻¹ M⁻¹ 92 18%98.3% 98.2%

5-(N-1-Benzylcarboxamide)-2′-deoxycytidine-5′-O-triphosphate (9a)

¹H NMR (300 MHz, D₂O): δ=8.45 (s, 1H), 7.25 (m, 5H), 6.14 (t, J=6.9 Hz,1H), 4.57 (m, J=2.9 Hz, 1H), 4.43 (dd, J=20.2, 15.4 Hz, 2H), 4.17 (m,3H), 2.39 (m, 1H), 2.27 (m, 1H). 13C NMR ³¹P NMR (300 MHz, D₂O): δ=−9.96(d, J=50.0 Hz, 1P), −11.43 (d, J=50.8 Hz, 1P), −23.24 (t, J=50.5 Hz, 1PMS m/z: [M⁻] calcd for C₁₇H₂₁N₄O₁₄P₃, 599.04; found, 599.1 [M]⁻.

5-(N-1-Naphthylmethylcarboxamide)-2′-deoxycytidine-5′-O-triphosphate(9b)

¹H NMR (500 MHz, D₂O): δ=8.12 (s, 1H), 7.98 (d, J=8.5 Hz, 1H), 7.69 (d,J=8.1 Hz, 1H), 7.58 (m, 1H), 7.40 (m, 1H), 7.33 (m, 1H), 7.24 (m, 1H),5.87 (t, J=6.7 Hz, 1H), 4.66 (d, J=8.1 Hz, 2H), 4.40 (m, J=3.0 Hz, 1H),4.04 (m, 3H), 2.21 (ddd, J=14.1, 6.0, 3.4 Hz, 1H), 2.06 (m, 1H). ¹³C NMR(500 MHz, D₂O): δ=165.95 (s, 1C), 163.15 (s, 1C), 155.22 (s, 1C), 143.33(s, 1C), 133.35 (s, 1C), 133.17 (s, 2C), 130.55 (s, 2C), 128.66 (s, 1C),127.84 (s, 1C), 126.65 (s, 1C), 126.13 (s, 1C), 125.64 (s, 1C), 125.12(s, 1C), 123.11 (s, 1C), 100.55 (s, 1C), 86.88 (s, 1C), 85.87 (d,J=55.95 Hz, 1C), 70.76 (s, 1C), 65.38 (d, J=36 Hz, 1C), 41.19 (s, 1C),39.61 (m, 1C). ³¹P NMR (500 MHz, D₂O): δ=−10.99 (d, J=82.4 Hz, 1P),−11.61 (d, J=84.9 Hz, 1P), −23.47 (t, J=83.5 Hz, 1P). MS nm/z: [M⁻]calcd for C₂₁H₂₄N₄O₁₄P₃, 649.36; found, 649.0 (ESI⁻).

5-(N-3-Phenylpropylcarboxamide)-2′-deoxycytidine-5′-O-triphosphate (9c)

¹H NMR (500 MHz, D₂O): δ=8.07 (s, 1H), 7.11 (m, 4H), 6.98 (m, 1H), 6.00(t, J=6.5 Hz, 1H), 4.44 (m, J=3.0 Hz, 1H), 4.06 (m, 3H), 3.21 (m, 1H),3.13 (m, 1H), 2.50 (t, J=7.5 Hz, 2H), 3.13 (ddd, J=14.1, 10.9, 3.1 Hz,1H), 2.13 (m, 1H), 1.76 (m, 2H). ¹³C NMR (500 MHz, D₂O): δ=165.85 (s,1C), 163.50 (s, 1C), 155.73 (s, 1C), 142.94 (s, 1C), 142.40 (s, 1C),128.55 (s, 2C), 128.40 (s, 2C), 125.72 (s, 1C), 101.15 (s, 1C), 86.93(s, 1C), 85.96 (d, J=55.2 Hz, 1C), 70.90 (s, 1C), 65.38 (d, J=37.6 Hz,1C), 39.88 (s, 1C), 39.55 (s, 1C), 32.74 (s, 1C), 26.68 (s, 1C). ³¹P NMR(500 MHz, D₂O): δ=−11.00 (d, J=82.7 Hz, 1P), −11.09 (d, J=85.7 Hz, 1P),−23.53 (t, J=84.3 Hz, 1P). MS m/z: [M⁻] calcd for C₁₉H₂₅N₄O₁₄P₃, 627.35;found, 627.0 (ESI⁻).

Example 5: Solid-Phase Oligonucleotide Synthesis

An ABI 3900 automated DNA synthesizer (Applied Biosystems, Foster City,Calif.) was used with conventional phosphoramidite methods with minorchanges to the coupling conditions for modified phosphoramidites (7a-c)(Table 4). Reagent (7a) was used as a 0.1 M solution indichloromethane/acetonitrile (1/1) and reagents (7b) and (7c) were usedas 0.1 M solutions in acetonitrile. Solid support was an ABI stylefritted column packed with controlled pore glass (CPG, Prime Synthesis,Aston, Pa.) loaded with 3′-DMT-dT succinate with 1000 Å pore size.Deprotection was accomplished by treatment with 20% diethylamine inacetonitrile followed by gaseous methylamine cleavage and deprotectionfor 2 hrs at 35° C. Identity and percent full length (% FL) product weredetermined on an Agilent 1290 Infinity with an Agilent 6130B singlequadrupole mass spectrometry detector using an Acquity OST C18 column1.7 μm 2.1×100 mm (Waters Corp., Milford, Mass.), using a gradient of 0to 25 percent B in 11 minutes (Buffer A: 100 mM1,1,1,3,3,3-hexafluoro-2-propanol, 8.6 mM triethylamine, pH 8.25; BufferB: 10% Buffer A in 90% acetonitrile).

TABLE 4 ABI 3900 Coupling Cycle Parameters (50 nmol scale) StepOperation Purpose reps Reagent volume, μl wait, sec pre Prep Supportwash 3 ACN 200 0 Prep Support detritylation 2 Deblock 50 0 1 Couplingcycle detritylation 3 Deblock 50 3 2 Coupling cycle wash 1 ACN 195 0 3(ATG) Coupling cycle coupling 2 Activator, amidite 36 + 19 30 + 175 3(7a-c) Coupling cycle coupling 3 Activator, amidite 36 + 19 60 + 250 4Coupling cycle capping 1 Cap A, B 15 + 15 5 5 Coupling cycle oxidation 1oxidizer 35 3 8 Coupling cycle wash 1 ACN 190 0 post Finalize Oligodetritylation 2 Deblock 140 0 Finalize Oligo wash 4 ACN 199 0 FinalizeOligo dry support 1 ACN; Ar 199 0 Key: ACN Acetonitrile Deblock 10%Dichloroacetic acid in toluene Activator 0.3M 5-Benzylmercaptotetrazoleand 0.5% N-methylimidazole in ACN Oxidizer 0.025M Iodine in 44.9%ACN/45% pyridine/10.1% water Cap A Acetic Anhydride in pyridine andtetrahydrofuran Cap B 1-Methylimidazole in tetrahydrofuran Ar Dry argonflush for 20 sec

Primer Extension Assay

The modified nucleoside triphosphates were evaluated as substrates forKOD exonuclease-minus DNA polymerase in a primer extension assay using astandard template that contained all possible triple nucleotidecombinations. The template sequence was:

(SEQ ID NO: 1) 5′-TTTTTTTTCTTCTTCTCCTTTCTCTTCCCAAAATCACACGGACCCAGGGCATTCTAGATATGGTTTACGCTCAAGCGAACTTGCCGTCCTGAGTGTAAAGAGGGAAAgagggcagggtgtggcatatatat-3′. (RC70X27.37, TriLink Biotechnologies)  The primer sequence was:(SEQ ID NO: 2) 5′-atatatatgccacaccctgccctc-3′.((AT)4-5P27, IDT Technologies) 

In brief, 10 pmoles of primer were labeled with 10 pmoles of ³²P-ATP at37° C. for 30 minutes with 3′ phosphatase minus T4 polynucleotide Kinase(New England Biolabs) in 7 mM Tris-HCl, pH 7.6 @ 25° C., 10 mM MgCl₂, 5mM dithiothreitol, and purified by passage through two Sephadex G-50cartridges (GE Healthcare). The 30 μL primer extension reactionscontained 120 mM Tris-HCl, pH 7.8, 10 mM KCl, 7 mM MgSO₄, 6 mM(NH₄)₂SO₄, 0.001% BSA, 0.1% Triton X-100, 3 pmoles of template, 6 pmolesof primer, and 7.5 Units of KOD exonuclease-minus DNA polymerase (EMDNovagen). The reactions were incubated at 96° C. for 30 seconds and 65°C. for 1 hour in a 96-well plate in an MJ thermocycler (Bio-Rad).

Five μL samples were analyzed on 8% acrylamide, 7 M urea, 1×TBE gels(Life Technologies) and exposed for 1 hour on an imaging plate beforescanning in a FujiFilm FLA3000 phosphorimager (GE Healthcare).

Example 6: Synthesis of Nucleic Acid Molecules withCytidine-5-Carboxamide Modified Nucleotides

This example provides the methods for making nucleic acid moleculeshaving cytidine-5-carboxamide modified nucleotides.

The CEP (phosphoramidite) reagents (7a-c; Scheme 1, Example 1) weretested for use solid-phase oligonucleotide synthesis on an automatedsynthesizer. For each new amidite reagent, six differentoligonucleotides varying in length from 34 to 39 nucleotides in length(or 34, 35, 36, 37, 38 or 39 nucleotides in length) were synthesizedwith an insertion of from 0, 1, 2, 3, 4 or 5 cytidine-5-carboxamidemodified nucleotides in consecutive internal positions, based on themodel sequence shown below. The “X” in the sequence indicates thelocation of the cytidine-5-carboxamide modified nucleotides within thesequence.

(SEQ ID NO: 3) 5′-GAGTGACCGTCTGCCTGX₀₋₅CAGACGACGAGCGGGA-3′

Table 5 below summarizes the percent yield of the oligonucleotidessynthesize with from 0 to 5 cytidine-5-carboxamide modified nucleotides.

TABLE 5 Synthetic DNA sequences Incorporating Modified 2′-DeoxycytidinesSequence HPLC LC/MS Data Cytidine Mod/ X_(n) Data FL Expected FLObserved (Phosphoramidite) n = % FL Mass (amu) Mass (amu) Δ (amu)Benzyl/ 0 65 10533.8 10531.2 2.6 (7a) 1 60 10955.1 10953.6 1.5 2 6411376.4 11375.4 1.0 3 65 11797.7 11797.9 0.2 4 52 12219.0 12220.2 1.2 552 12640.4 12642.5 2.1 1-Naphthylmethyl/ 0 60 10533.8 10531.3 2.5 (7b) 167 11005.1 11003.3 1.7 2 64 11476.4 11475.8 0.6 3 54 11947.7 11948.2 0.54 47 12419.0 12420.2 1.2 5 49 12890.3 12892.9 2.6 3-Phenylpropyl/ 0 6910533.8 10531.3 2.4 (7c) 1 59 10983.1 10981.3 1.8 2 68 11432.5 11431.60.9 3 41 11881.9 11882.0 0.1 4 42 12331.2 12332.2 1.0 5 49 12780.612782.8 2.2

The results indicate that full-length synthetic yields for 1 to 3sequential couplings of the modified cytidine phosphoramidites (7a-c)were comparable to unmodified DNA phosphoramidites. Some loss of yieldwas observed for 4 or 5 sequential couplings of modified cytidines;however significant amounts of full length product were obtained andconfirmed in all cases.

Example 7: Incorporation of Triphosphate Reagents (TPP Reagent) ofCytidine-5-Carboxamide Modified Nucleotides by KOD DNA Polymerase

This example shows that the cytidine-5-carboxamide modified nucleotides(9a, 9b and 9c of Scheme 2) may be used as substrates by the KODexonuclease-minus DNA polymerase.

FIG. 1 below shows the results of a primer extension assay. All threemodified cytidine triphosphates were incorporated at least asefficiently as natural, unmodified 2′-deoxycytidine in this assay.

In summary, a practical process for synthesizing cytidine-5-carboxamidemodified nucleotides as both 5′-O-triphosphates and 3′-O-CEPphosphoramidites provides valuable new reagents for in vitro selectionand post-SELEX optimization of aptamers.

Example 8: Selection of Cytidine-5-Carboxamide Modified NucleotideAptamers with SELEX

This example shows that cytidine-5-carboxamide modified nucleotideaptamers may be selected for binding to a protein target with SELEX.Further, this example shows a comparison of cytidine-5-carboxamidemodified nucleotide aptamers derived from SELEX to a specific proteintarget to uridine-5-carboxamide modified nucleotide aptamers derivedfrom SELEX to the same protein target.

Four different proteins were used as targets for SELEX: PCSK9, PSMA,ERBB2 and ERBB3.

Modified random libraries were enzymatically synthesized using KOD DNAPolymerase using standard oligonucleotide synthesis protocol. The randomlibraries included a control library labeled as “dC/dT” and contained noC-5 modified nucleotides; a NapdC library, a NapdU(5-[N-(1-naphthylmethyl)carboxamide]-2′-deoxyuridine) library, a PPdClibrary and a PPdU (5-[N-(phenyl-3-propyl)carboxamide]-2′-deoxyuridine)library. All random libraries were enzymatically synthesized using thesesame conditions targeting at least 5 nmole final product per library(50-60% yield from starting antisense template). The crude librarieswere concentrated using 10 kDa NMW cut-off ultrafiltration centrifugaldevices. The concentrated product was spun down to remove anystreptavidin (SA) agarose bead using SPIN-X microcentrifuge tubes, andquantified by measuring absorbance at 260 nm and using estimatedabsorbance coefficient. Each modified sense library was qualitycontrolled for its inability to shift free SA for contamination ofbiotinylated anti-sense strands and also standard PCR amplificationconditions (data not shown).

Recombinant human PCSK9 protein Gln31-Gln692 (75.1 kDa) with C-terminalpoly His tag and produced in human 293 (HEK293) cells was obtained fromACRO Biosystems (Cat# PC9-H5223). This protein is glycosylated andauto-proteolytically cleaved DTT-reduced protein runs as 20 KDa(prodomain) and 62 kDa (mature secreted protein) polypeptides onSDS-PAGE gel.

Recombinant human PSMA (˜110 kDa) was obtained from R&D Systems(Cat#4234-ZN-010) which was CHO-derived Lys44-Ala750 with N-terminal6×His tag.

Recombinant human ErbB2 protein Thr23-Thr652 (72.4 kDa) with C-terminalpoly His tag and produced in human 293 (HEK293) cells was obtained fromACRO Biosystems (Cat# HE2-H5225). As a result of glycosylation,DTT-reduced protein migrates at 90-110 kDa range on SDS-PAGE for thistarget.

Recombinant human ErbB3 protein Ser20-Thr643 (71.5 kDa) with C-terminal6×His tag and produced in human 293 (HEK293) cells was obtained fromACRO Biosystems (Cat# ER3-H5223). As a result of glycosylation,DTT-reduced protein migrates at 100-110 kDa range on SDS-PAGE for thistarget.

All the targets used in SELEX were checked for their purity andpartition capture efficiency using magnetic Dynabeads® His-Tag capturebeads Life technologies (Cat#10104D). All the targets were efficientlycaptured using His tag capture beads.

The SELEX protocol (5 mM DxSO4 Kinetic Challenge starting Round 2) wasfollowed for all the selection steps. For round one, 1000 pmole (˜10¹⁵sequences) random library for each SELEX experiment was used. Targetswere at 50 pmole concentration (captured on 500 μg His Capture Beads)and complexes were equilibrated at 37° C. for 1 hr. and then washedseveral times with 1×SB 18, 0.05% TWEEN20. Selected sequences wereeluted with 20 mM NaOH, neutralized and PCR amplified using 5′ OH primerand 3′ biotinylated primer. The amplified double-stranded unmodified DNAwas captured on SA magnetic beads, washed and sense DNA was eluted off,and primer extended using modified nucleotides to regenerate enrichedmodified pool to be used in next round of SELEX experiments.

A total of six selection rounds were completed. In general, samples wereat 1 nM protein concentrations as C_(t) differences for +/−proteinselection samples were not improving indicating probably no furtherenrichment of sequences, SELEX was stopped at Round 6, modified senseeDNA was made for all samples and pool affinities to respective targetswere performed. It should be noted that unmodified control DNA enrichedlibraries were processed in the similar manner to modified libraries,even though PCR amplified and eluted sense DNA strand could be directedused in next SELEX rounds.

The eDNAs were radio-labeled and filter binding assays were performedfor all enriched pools and compared with corresponding starting randomlibraries. Random libraries did not bind to the four protein. Table 6shows the affinity data results for the four protein targets PCSK9,PSMA, ERBB2 and ERBB3.

TABLE 6 Target Pool Affinity Data After 6 Rounds of SELEX Pool Affinity(nM) Protein Target NapdC NapdU PPdC PPdU PCSK9 1.57 nM 0.72 nM 2.44 nM1.02 nM PSMA 0.86 nM  7.8 nM 6.32 nM 6.79 nM ERBB2 11.4 nM 10.2 nM 71.3nM 6.57 nM ERBB3 0.25 nM 0.38 nM 15.9 nM  0.3 nM

As shown in Table 6, the average binding affinity (K_(d)) for a pool ofnucleic acid aptamers having at least one C-5 modified cytodinenucleotide that was enriched for binding to the PSMA target protein viaSELEX was 0.86 nM (NapdC), compared to 7.8 nM for a pool of nucleic acidaptamers having a NapdU against the same protein; and 6.32 nM (PPdC),compared to 6.79 nM for a pool of nucleic acid aptamers having a PPdUagainst the same protein. The average binding affinity (K_(d)) for apool of nucleic acid aptamers having at least one C-5 modified cytodinenucleotide that was enriched for binding to the ERBB3 target protein viaSELEX was 0.25 nM (NapdC), compared to 0.38 nM for a pool of nucleicacid aptamers having a NapdU against the same protein.

Further analysis of the SELEX pools of nucleic acid aptamers enrichedfor binding to the proteins PSCK9, PSMA, ERBB2 and ERBB3 showed thatSELEX performed with nucleic acid aptamers having at least one C-5modified cytodine nucleotide provided a greater number of multicopy(greater than two (2) copies) nucleic acid sequences in comparison toSELEX performed with nucleic acid aptamers having at least one C-5modified uridine nucleotide. Table 7 below summarizes the differences ofSELEX performed with NapdC, NapdU, PPdC and PPdU.

TABLE 7 Number of Multicopy (>2) Sequenes After 6 Rounds of SELEX Numberof Multicopy (>2) Sequences Protein Target NapdC NapdU PPdC PPdU PCSK9151 78 302 46 PSMA 187 143 251 58 ERBB2 52 65 85 40 ERBB3 144 160 94 30

In general, Table 7 shows that the C-5 modified cytodine nucleotideprovides a greater number of sequences having more than two copies inthe pool of nucleic acid aptamer sequences enriched for target proteinbinding via SELEX. Thus, in general, the C-5 modified cytodinenucleotide in SELEX against a protein target provides a greaterdiversity of multicopy nucleic acid sequences, which consequentlyprovides a greater number of nucleic acid aptamers to select for furthercharacterization and development as a protein binding reagent and/ortherapeutic. This benefit C-5 modified cytodine nucleotides is betterrealized in light of the challenges associated with developing a nucleicacid aptamer for a particular purpose (e.g., protein binder forassays—pull-down assays, protein purification, mass spectroscopy; areagent tool and/or therapeutic—protein agonists or antagonist). Thegreater number of multicopy nucleic acid aptamer provides a greaternumber of sequences that may be screened and further developed for aparticular purpose and reduce the failure rate of such development.

REFERENCES

-   Gold, L. et al. (2010) Aptamer-based proteomic technology for    biomarker discovery. PLoS ONE, 5(12), e15004.-   Hollenstein, M. (2012) Synthesis of Deoxynucleoside Triphosphates    that Include Proline, Urea, or Sulfonamide Groups and Their    Polymerase Incorporation into DNA. Chemistry, A European Journal,    18, 13320-13330.-   Imaizumi, Y. et al. (2013) Efficacy of Base-Modification on Target    Binding of Small Molecule DNA Aptamers. J. Am. Chem. Soc., 135(25),    9412-9419.-   Davies, D. R. et al. (2012) Unique motifs and hydrophobic    interactions shape the binding of modified DNA ligands to protein    targets. PNAS, 1 90(49), 19971-19976.-   Lee, K. Y. et al. (2010) Bioimaging of Nucleolin Aptamer-Containing    5-(N-benzylcarboxamide)-2′-deoxyuridine More Capable of Specific    Binding to Targets in Cancer Cells. J. Biomedicine and    Biotechnology, article ID 168306, 9 pages.-   Kerr, C. E. et al. Synthesis of N,N-Dialkylaniline-2′-deoxyuridine    Conjugates for DNA-Mediated Electron Transfer Studies. Nucleosides,    Nucleotides & Nucleic Acids, 19(5&6), 851-866.-   Gaballah, S. T. et al. (2002) Synthesis of 5-(2,2′-Bipyridyl- and    2,2′-Bipyridinediiumyl)-2′-deoxyuridine Nucleosides: Precursors to    Metallo-DNA Conjugates. Nucleosides, Nucleotides & Nucleic Acids    21(8&9), 547-560.-   Vaught, J. D. et al. (2004) T7 RNA Polymerase Transcription with    5-Position Modified UTP Derivatives. J. Am. Chem. Soc., 126,    11231-11237.-   Vaught, J. D.; et al. (2010) Expanding the chemistry of DNA for in    vitro selection. J. Am. Chem. Soc., 132(12), 4141-4151.-   Nomura, Y. et al. (1997) Site-specific introduction of functional    groups into phosphodiester oligodeoxynucleotides and their thermal    stability and nuclease-resistance properties. Nucleic Acids Res.,    25(14), 2784-2791.-   Nomura, Y. et al. (1996) Nucleosides and Nucleotides. 161.    Incorporation of 5-(N-aminoalkyl)carbamoyl-2′-deoxycytidines into    oligodeoxynucleotides by a convenient post-synthetic modification    method. Bioorganic & Medicinal Chemistry Letters, 6(23), 2811-2816.-   Uozumi, Y. et al. (2001) Double Carbonylation of Aryl Iodides with    Primary Amines under Atmospheric Pressure Conditions Using the    Pd/Ph₃P/DABCO/THF System. J. Org. Chem. 66, 5272-5274.-   Takacs, A. et al. (2008) Palladium-catalyzed Aminocarbonylation of    Iodoarenes and Iodoalkenes with Aminophosphonate as N-Nucleophile.    Tetrahedron. 64, 8726-8730.-   Ross, B. S. et al. (2006) Efficient Large-Scale Synthesis of    5′-O-Dimethoxytrityl-N4-Benzoyl-5-methyl-2′-deoxycytidine.    Nucleosides, Nucleotides & Nucleic Acids, 25, 765-770.-   Sanghvi, Y. S. et al. (2000) Improved Process for the Preparation of    Nucleosidic Phosphoramidites Using a Safer and Cheaper Activator.    Organic Process Research & Development, 4, 175-181.-   Still, W. C. et al. (1978) Rapid Chromatographic Technique for    Preparative Separations with Moderate Resolution. J. Org. Chem., 43,    2923-2925.-   Leonard, N. J. and Neelima (1995) 1,1,1,3,3,3-Hexafluoro-2-propanol    for the Removal of the 4,4′-Dimethoxytrityl Protecting Group from    the 5′-Hydroxyl of Acid-Sensitive Nucleosides and Nucleotides.    Tetrahedron Letters, 36(43), 7833-7836.-   Ludwig, J. and Eckstein, F. (1989) Rapid and Efficient Synthesis of    Nucleoside 5′-O-(1-Thiotriphosphates), 5′-Triphosphates and    2′,3′-Cyclophosphorothioates Using    2-Chloro-4H-1,3,2-benzodioxaphophorin-4-one. J. Org. Chem., 54,    631-635.-   Ito, T. et al. (2003) Synthesis, thermal stability and resistance to    enzymatic hydrolysis of the oligonucleotides containing    5-(N-aminohexyl)carbamoyl-2′-O-methyluridines. Nucleic Acids Res.,    31(10), 2514-2523.

The invention claimed is:
 1. A compound comprising the structure shown in Formula I:

wherein R is independently a —(CH₂)_(n)—, wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; R^(X1) comprises

* denotes the point of attachment of the R^(X1) group to the —(CH₂)_(n)— group; and R^(X4) is independently selected from the group consisting of a branched or linear lower alkyl (C1-C20); a hydroxyl group; F, Cl, Br, I; nitrile (CN); boronic acid (BO₂H₂); carboxylic acid (COOH); carboxylic acid ester (COOR^(X2)); primary amide (CONH₂); secondary amide (CONHR^(X2)); tertiary amide (CONR^(X2)R^(X3)); sulfonamide (SO₂NH₂); N-alkylsulfonamide (SONHR^(X2)); R^(X2) and R^(X3) are independently, for each occurrence, selected from the group consisting of a branched or linear lower alkyl (C1-C20); phenyl (C₆H₅); an R^(X4) substituted phenyl ring (R^(X4)C₆H₄), wherein R^(X4) is defined above; a carboxylic acid (COOH); a carboxylic acid ester (COOR^(X5)), wherein R^(X5) is a branched or linear lower alkyl (C1-C20); and cycloalkyl, wherein R^(X2)=R^(X3)=(CH₂)_(n); X is independently selected from the group consisting of —H, —OH, —OMe, —O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃ and -azido; R′ is independently selected from the group consisting of a —H, —OAc; —OBz; —P(NiPr₂)(OCH₂CH₂CN); and —OSiMe₂tBu; R″ is independently selected from the group consisting of a hydrogen, 4,4′-dimethoxytrityl (DMT) and triphosphate (—P(O)(OH)—O—P(O)(OH)—O—P(O)(OH)₂) or a salt thereof; and salts thereof.
 2. The compound of claim 1, wherein R^(X4) is independently selected from the group consisting of a branched or linear lower alkyl (C1-C6); a —OH; a —F and carboxylic acid (COOH).
 3. The compound of claim 1, wherein X is independently selected from the group consisting of —H, —OMe and —F.
 4. The compound of claim 1, wherein R′ is selected from the group consisting of a —H, —OAc and —P(NiPr₂)(OCH₂CH₂CN).
 5. The compound of claim 1, R″ is a triphosphate (—P(O)(OH)—O—P(O)(OH)—O—P(O)(OH)₂).
 6. The compound of claim 2, Wherein n is 0, 1, 2 or
 3. 7. A nucleic acid molecule comprising the compound of claim
 1. 8. A method for making a compound having Formula I:

wherein R is independently a —(CH₂)_(n)—, wherein n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; R^(X1) is independently selected from the group consisting of

* denotes the point of attachment of the R^(X1) group to the —(CH₂)_(n)— group; R^(X4) is independently selected from the group consisting of a branched or linear lower alkyl (C1-C20); a hydroxyl group; F, Cl, Br, I; nitrile (CN); boronic acid (BO₂H₂); carboxylic acid (COOH); carboxylic acid ester (COOR^(X2)); primary amide (CONH₂); secondary amide (CONHR^(X2)); tertiary amide (CONR^(X2)R^(X3)); sulfonamide (SO₂NH₂); N-alkylsulfonamide (SONHR^(X2)); R^(X2) and R^(X3) are independently, for each occurrence, selected from the group consisting of a branched or linear lower alkyl (C1-C20); phenyl (C₆H₅); an R^(X4) substituted phenyl ring (R^(X4)C₆H₄), wherein R^(X4) is defined above; a carboxylic acid (COOH); a carboxylic acid ester (COOR^(X5)), wherein R^(X5) is a branched or linear lower alkyl (C1-C20); and cycloalkyl, wherein R^(X2)=R^(X3)=(CH₂)_(n); X is independently selected from the group consisting of —H, —OH, —OMe, —O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃ and -azido; R′ is independently selected from the group consisting of a —H, —OAc; —OBz; —P(NiPr₂)(OCH₂CH₂CN); and —OSiMe₂tBu; R″ is independently selected from the group consisting of an hydrogen, 4,4′-dimethoxytrityl (DMT) and triphosphate (—P(O)(OH)—O—P(O)(OH)—O—P(O)(OH)₂) or a salt thereof; the method comprising providing a compound having Formula IX

wherein, R^(X6) is an iodine or bromine group; R^(X7) and R^(X8) axe independently, for each occurrence, a hydrogen or protecting group; X is independently selected from the group consisting of —H, —OH, —OMe, —O-allyl, —F, —OEt, —OPr, —OCH₂CH₂OCH₃ and -azido; and transforming the compound having Formula IX by a palladium(0) catalyzed reaction in the presence of R^(X1)—R—NH₂, carbon monoxide and a solvent; and isolating the compound having Formula I.
 9. The method of claim 8, wherein R^(X4) is independently selected from the group consisting of a branched or linear lower alkyl (C1-C6); a —OH; a —F and carboxylic acid (COOH).
 10. The method of claim 8, wherein R′ is selected from the group consisting of a —H, —OAc and —P(NiPr₂)(OCH₂CH₂CN).
 11. The method of claim 8, R″ is a hydrogen or triphosphate (—P(O)(OH)—O—P(O)(OH)—O—P(O)(OH)₂); or salt thereof.
 12. The method of claim 8, wherein n is 1, 2 or
 3. 13. The method of claim 8, wherein the protecting group is selected from the group consisting of triphenylmethyl, p-anisyldiphenylmethyl, di-p-anisyldiphenylmethyl, p-dimethoxy trityltrityl, formyl, t-butyloxycarbonyl, benzyloxycarbonyl, 2-chlorobenzyloxycarbonyl, 4-chlorobenzoyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl, furfurylcarbonyl, t-amyloxycarbonyl, adamantyloxycarbonyl, 2-phenylpropyl-(2)-oxycarbonyl, 2-(4-biphenyl)propyl-(2)-oxycarbonyl, 2-nitrophenylsulfenyl and diphenylphosphinyl. 